METHODS FOR THE DETECTION OF CLOSTRIDIUM BOTULINUM AND ITS

NEUROTOXINS

In view of the danger presented by C. botulinum and its potent neurotoxins, practical work is restricted to containment laboratories offering an appropriate degree of protection. It must be ensured that all appropriate safety guidelines are in place before work is started.

Cultural Methods for Proteolytic C. botulinum and Nonproteolytic C. botulinum

Culture of C. botulinum may be required for studies of the extent of contamination of the

may not be specifi c for neurotoxins (Sakaguchi, 1979;

Huhtanen et al., 1992; Potter et al., 1993); and (iv) many tests require complex and expensive ampli-fi cation systems to achieve the sensitivity of the mouse test (e.g., Shone et al., 1985; Modi et al., 1986; Doell-gast et al., 1993). Despite these limitations, ELISAs and other immunochemical methods have been used widely for detection of C. botulinum neurotoxins (Table 9). An ELISA developed by Ferreira and col-leagues (Ferreira and Crawford, 1998; Ferreira, 2001;

Ferreira et al., 2001, 2004) offers independent detec-tion of types A, B, E, and F neurotoxins and has been used to detect neurotoxin in food associated with botulism outbreaks in the United States (Ferreira et al., 2001, 2004). The test is not as sensitive as the mouse test. Sharma et al. (2006) reported on an amplifi ed ELISA that detected 5 50% minimum lethal doses (MLD50)/ml of type A, B, E, and F neuro-toxins in casein buffer and 60 MLD50/ml in various foods. A simple, rapid, cheap, sensitive, chemilumi-nescent slot blot immunoassay has been used for the quantifi cation of type E neurotoxin (Cadieux et al., 2005). Lateral fl ow immunoassays are extremely easy to use and provide a very rapid detection of the tar-get, typically in 15 to 30 min (Aldus et al., 2003;

Capps et al., 2004; Sharma et al., 2005). These assays are qualitative and appear to show promise only as an initial screening tool for botulinum neurotoxin, due to their relatively high detection limit (1,000 to 2,000 MLD50/ml for type A, B, and F neurotoxins [Sharma et al., 2005; Gessler et al., 2007]). A further method uses paramagnetic bead-based electrochemi-luminescence to detect botulinum neurotoxins. In this test the signal is generated after capture, by a magnet, of complexes containing paramagnetic beads complexed with capture and detection antibodies and botulinum neurotoxin. The test permits rapid throughput of samples, requiring 2 hours to com-plete. Detection limits for type A, B, E, and F neuro-toxins ranged from 2 to 70 MLD50/ml in clinically relevant matrices and 1 to 70 MLD50/ml in selected food matrices (Rivera et al., 2006). A number of other immunochemical methods have been developed for quantifi cation of botulinum neurotoxins, such as fi ber-optic evanescent-wave immunosensors (Scar-latos et al., 2005; Sharma and Whiting, 2005;

Lindström and Korkeala, 2006). Colony immuno-blot techniques have been used to identify colonies of proteolytic C. botulinum types A and B (Goodnough et al., 1993) and nonproteolytic C. botulinum type E (Dezfulian, 1993; Goodnough et al., 1993).

A number of in vitro assays have been developed that quantify the highly specifi c endopeptidase activ-ity of the botulinum neurotoxin light chains. The fi rst assays to be developed used an immunochemical is a favored nonselective solid medium, as colonies of

proteolytic C. botulinum and nonproteolytic C. botu-linum have a typical appearance associated with their lipase activity. Selective plating media include C. bot-ulinum isolation agar (Dezfulian et al., 1981) and botulinum selective medium (Mills et al., 1985). The trimethoprim in these media may inhibit nonprote-olytic C. botulinum (Hatheway, 1988).

Detection and Quantifi cation of C. botulinum Neurotoxins

It may be necessary to test for botulinum neuro-toxin in samples of food, clinical samples, or enrich-ment media. The standard method for detection and identifi cation of botulinum neurotoxin has been intraperitoneal injection into mice (Hatheway, 1988;

Solomon and Lilly, 2001). Specifi city is achieved by the use of specifi c antisera and by observations of typical symptoms of botulism in the mice prior to death. It is necessary to treat samples with trypsin to detect neurotoxin formed by nonproteolytic C. botu-linum. Trypsin is required to convert the single-chain neurotoxin to the more toxic dichain form. The mouse test is extremely sensitive (5 to 10 pg of toxin), measures the biological activity of the neuro-toxin, and is both repeatable and reproducible (Kautter and Solomon, 1977; Hatheway, 1988).

Additionally, there is the potential to detect previ-ously undescribed neurotoxins, atypical neurotoxins, and antigenic variants. Disadvantages of the mouse test are the ethical issues concerning the use of ani-mals, the need to wait several days before a sample can be judged negative, the expense, and the need for skilled personnel. A number of alternative animal methods have been described, some of which are nonlethal (Sesardic et al., 1996; Pearce et al., 1997).

Immunochemical methods for the detection of botulinum neurotoxins include enzyme-linked immu-nosorbent assays (ELISA), lateral fl ow immunoas-says, and various other tests (Scarlatos et al., 2005;

Sharma and Whiting, 2005; Lindström and Korkeala, 2006). These methods are generally cheaper and much easier to use than the mouse test, and some have the same sensitivity and specifi city. The various immunochemical methods may be subject to various limitations, however; for example, (i) some may react differently with neurotoxins of a specifi c type produced by different strains, since these neuro-toxins may differ in antigenicity (Gibson et al., 1987, 1988; Huhtanen et al., 1992; Doellgast et al., 1993;

Ekong et al., 1995; Smith et al., 2005); (ii) some may react with biologically inactive neurotoxin; (iii) some use antibodies that were raised to preparations containing a mixture of antigens, so that the tests

assay for type B neurotoxin has been developed that captures the neurotoxin on an immunoaffi nity col-umn or microtiter plate, prior to an endopeptidase assay (Wictome and Shone, 1998; Wictome et al., 1999a, 1999b). This method worked well with foods including meat, fi sh, and cheese and was able to dis-tinguish between type B neurotoxin formed by prote-olytic C. botulinum and nonproteprote-olytic C. botulinum.

Alternative assays employ a synthetic peptide (to mimic SNAP-25 or VAMP) labeled with quenched fl uorophores as the substrate. The endopeptidase activity of the botulinum neurotoxins leads to cleav-age of these peptides and a release of fl uorescence (Schmidt et al., 2001; Schmidt and Stafford, 2003).

The endopeptidase activity of botulinum neurotoxins has also been quantifi ed using mass spectrometry.

Synthetic substrates are incubated with the test sam-ple, and the product peptides identifi ed on the basis of their mass. The concentrations of all seven neuro-toxin types were measured simultaneously in a single test sample, and with samples in buffer, this assay was more sensitive than the mouse test (Barr et al., 2005; Boyer et al., 2005). To avoid problems encoun-tered with nonspecifi c proteases, this method has been expanded to include an antibody capture method approach to detect cleavage of SNAP-25 or VAMP

by botulinum neurotoxins and were often as sensitive as the mouse test. In these assays, a fragment of the target protein (SNAP-25 or VAMP) is attached to a microtiter plate and serves as the substrate. The sam-ple containing neurotoxin is then added, and after a period of incubation, specifi c antibodies are added that bind to the cleaved target protein and then sec-ondary antibodies and a detection system are added.

Advantages over ELISA procedures follow: (i) the tests measure the biological activity of the neurotoxin light chain (but not the heavy chain); (ii) variations in the antigenicity of neurotoxins of a specifi c type do not infl uence the response; and (iii) the problem resulting from antibodies having been raised to a mixture of antigens is eliminated. Endopeptidase assays for type A neurotoxin and type B neurotoxin were specifi c, did not cross-react with each other or other neurotoxins, and after signal amplifi cation, had detection limits of 30 to 40 MLD50/ml (Hallis et al., 1996). A highly sensitive endopeptidase assay devel-oped for therapeutic preparations of type A neuro-toxin correlated well with the mouse test and had a detection limit of 0.2 to 1.0 MLD50/ml (Ekong et al., 1997). In a slightly different format, a highly sensitive

Table 9. Examples of sensitive quantitative immunochemical methods for detection of C. botulinum neurotoxins Neurotoxins detected

(MLD50/ml) Comments Reference(s)

A (5–10) ELISA. Failed to detect neurotoxin produced by one type A strain. No cross-reaction with other clostridia, denatured neurotoxin, or other neurotoxin types. Complex amplifi cation system. Used with foods.

Shone et al. (1985), Gibson et al. (1987)

B (20) ELISA. Failed to detect neurotoxin produced by one type B strain. No cross-reaction with other clostridia or other neurotoxin. Used with foods. Complex amplifi cation system.

Modi et al. (1986), Gibson et al. (1988)

A (1–32), B (1–16) ELISA. May respond to antigens with no neurotoxicity. Correlation between the response from ELISA and mouse test not always consistent. Used with foods.

Huhtanen et al. (1992)

A (9), B (1), E (1) ELISA. Cross-reaction with other clostridia. Used extensively to measure formation of type A, B, and E neurotoxin in meat and in vegetable preparations. Also reacted with type F neurotoxin.

Potter et al. (1993), Carlin and Peck (1995), Fernandez and Peck (1999), Stringer et al.

(1999) A (1), B (1), E (1) ELISA. Weak reaction with neurotoxin from some strains. Used to

measure neurotoxin production by nonproteolytic C. botulinum in fi sh fi llets. Complex amplifi cation system.

Doellgast et al. (1993, 1994), Roman et al. (1994)

A (1–20) ELISA. Developed for therapeutic preparations. Ekong et al. (1995)

E (1–10) ELISA. No cross-reaction with other neurotoxins or other clostridia.

Used with foods.

Wong (1996) A (10), B (10), E (10), F (10) ELISA. Tested in a ring trial. Used to quantify neurotoxin present in

food samples associated with botulism outbreaks.

Ferreira and Crawford (1998), Ferreira (2001), Ferreira et al.

(2001, 2004) E (4) Chemiluminescent slot blot immunoassay. Used with bacterial cultures,

naturally contaminated soil, inoculated fi sh. Some cross-reaction.

Cadieux et al. (2005) A (2), B (2), E (5), F (1) ELISA. Low detection limit in casein buffer, higher in food samples

(60 MLD50/ml). High specifi city.

Sharma et al. (2006) A (50), B (70), E (5), F (1) Paramagnetic bead-based electrochemiluminescence. Sensitive and

specifi c detection in food and clinically relevant matrices.

Rivera et al. (2006)

further hybridization onto a polyester cloth mem-brane coated with cDNA probes to the PCR products.

In some of these tests, retargeting of the PCR primers may be required in order to ensure the detection of all recently described neurotoxin subtypes (Smith et al., 2005). There is also merit in confi rming the specifi city of a positive PCR result, for example, by sequencing of the PCR product. The use of molecular methods for the detection of C. botulinum has been reviewed recently (Lindström and Korkeala, 2006).

A number of typing tools have been used for the molecular characterization of strains of C. botuli-num. These include ribotyping, pulsed-fi eld gel elec-trophoresis (PFGE), DNA sequencing, DNA microarrays, PCR-based methods (e.g., repetitive ele-ment sequence-based PCR, amplifi ed fragele-ment length polymorphism [AFLP], and randomly amplifi ed poly-morphic DNA analysis), and focal plane array-Fourier transform infrared spectroscopy. Ribotyping is based on analysis of conservative ribosomal genes and is widely used for molecular typing of unknown bacteria and distinguished strains of proteolytic C. botulinum and nonproteolytic C. botulinum (Lind-ström and Korkeala, 2006). For PFGE, genomic DNA that has been digested with rare-cutting restriction enzymes is separated by electrophoresis. In a recent study, this technique was used to investigate the diver-sity of 55 strains of proteolytic C. botulinum (Nevas et al., 2005c). PFGE has a higher discriminatory power than ribotyping (Lindström and Korkeala, 2006). The genome sequence of proteolytic C. botuli-num strain Hall A (ATCC 3502, NCTC 13319) has now been published (Sebaihia et al., 2007), and the sequencing of genomes of other strains of proteolytic C. botulinum and nonproteolytic C. botulinum is in progress. Comparative genomic indexing using a DNA microarray based on the Hall A genome sequence was an effective tool to discriminate strains of proteolytic C. botulinum but not nonproteolytic C.

botulinum (Sebaihia et al., 2007). Unlike other typing methods, information is also provided on the genome content of the tested strains. It was found that 87%

to 96% of the Hall A presumptive genes were pos-sessed by nine other strains of proteolytic C. botuli-num and that 84% to 87% of the presumptive genes were shared with two strains of C. sporogenes. Two prophages present in the Hall A strain were absent in the 11 test strains. For repetitive element sequence-based PCR, PCR is targeted at conservative repetitive extragenic elements. A species-specifi c fi ngerprint can be derived from the size and number of amplifi cation products, while strain-specifi c differentiation is lim-ited. Hyytiä et al. (1999) used this method to charac-terize strains of proteolytic C. botulinum and nonproteolytic C. botulinum. For AFLP, genomic to partially purify and concentrate neurotoxin (e.g.,

from serum or stool), prior to quantifi cation of endo-peptidase activity (Kalb et al., 2006). Detection limits for samples spiked with type A, B, E, and F neurotox-ins were 1 to 20 MLD50/ml for human serum sam-ples and 1 to 200 MLD50/ml for stool samples. The entire method could be performed in 4 hours, but limitations are the high equipment costs and the need for highly trained personnel. Potential limitations of all endopeptidase activity tests include (i) interference by other proteases (although in some tests this has been addressed by capture of the neurotoxin prior to measurement of endopeptidase activity) and (ii) posi-tive reaction with neurotoxin that is inacposi-tive in vivo, (because the endopeptidase assays relate to the bio-logical activity of the light chain and would not be affected by inactivation of the heavy chain).

Molecular Methods for the Detection and Characterization of C. botulinum

A number of tests have been developed that use PCR to detect neurotoxin genes. While these tests do not detect the neurotoxin, they have been shown to be of considerable utility in the investigation of botu-lism outbreaks and in the testing of enrichment media during surveys for the presence of C. botuli-num in food, clinical, and environmental samples.

Following a cultural enrichment, these methods cor-related well with tests for neurotoxin using mice and, with the inclusion of a most-probable-number series of dilutions in the cultural enrichment, have been used for quantitative detection of bacteria contain-ing these genes in relevant samples (e.g., Hielm et al., 1996, 1998; Aranda et al., 1997; Lindström et al., 2001; Carlin et al., 2004; Nevas et al., 2005b; Myl-lykoski et al., 2006; Lindström and Korkeala, 2006;

Merivirta et al., 2006). The use of a cultural enrich-ment ensures good sensitivity, while minimizing pos-sible problems due to the presence of extracellular DNA or dead bacteria. Probes have been constructed that enable PCR tests for the nonspecifi c detection of all botulinum neurotoxin genes (Campbell et al., 1993) and for the specifi c detection of genes encod-ing each neurotoxin (e.g., Szabo et al., 1993; Fran-ciosa et al., 1994; Fach et al., 1995; Ferreira and Hamdy, 1995; Alsallami and Kotlowski, 2001; Wu et al., 2001; Fach et al., 2002; Carlin et al., 2004;

Lindström and Korkeala, 2006). An important step forward has been the development of multiplex PCR methods for simultaneous detection of type A, B, E, and F neurotoxin genes in a single reaction (Lind-ström et al., 2001, 2006b; Gauthier et al., 2005).

Detection in these tests was achieved by gel electro-phoresis confi rmation of product size or by

introduction of C. botulinum or its neurotoxin into the food chain through a bioterrorism act.

Acknowledgments. I am grateful for funding from the Competitive Strategic Grant of the BBSRC and other funders of the Institute of Food Research.

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DNA that has been digested with a pair of restriction enzymes is ligated with restriction site-specifi c adap-tors, and a subset of the fragments amplifi ed by PCR.

AFLP has been used to characterize 33 strains of pro-teolytic C. botulinum and 37 strains of nonpropro-teolytic C. botulinum (Keto-Timonen et al., 2005). This method clearly differentiated between proteolytic C. botulinum and nonproteolytic C. botulinum and was suitable for typing at the strain level. Randomly amplifi ed polymorphic DNA analysis involves PCR amplifi cation with randomly annealing univer-sal primers under conditions of low stringency.

This method and ribotyping, however, were not as effective as PFGE in discriminating strains of nonpro-teolytic C. botulinum type E from the Canadian arctic (Leclair et al., 2006). Focal plane array-Fourier transform infrared spectroscopy has been used for whole-organism fi ngerprinting of 44 strains of pro-teolytic C. botulinum and nonpropro-teolytic C. botuli-num (Kirkwood et al., 2006). This method provided rapid discrimination of the two C. botulinum groups.

CONCLUDING REMARKS

C. botulinum organisms are a heterogeneous group of bacteria that forms the botulinum neuro-toxin, the most potent substance known. Since spores of C. botulinum are ubiquitous in food, albeit often at a low concentration, it is necessary to apply treat-ments that destroy spores or store the food under con-ditions that prevent growth and neurotoxin formation.

In view of the severity of food-borne botulism, vigi-lance is needed to ensure that C. botulinum does not become an emerging pathogen. Nonproteolytic C.

botulinum has been identifi ed as the principal safety hazard in minimally heated refrigerated foods, and it is essential that research continues to underpin the safe development of these novel foods. It is important that as new food processes are applied (e.g., high hydrostatic pressure), the principle of equivalence with existing safe processes is adopted. The increased movement of raw materials and foods, through glo-balization of trade, may also bring an increased risk.

For example, three of the four food-borne botulism outbreaks reported in the United Kingdom between 2002 and 2005 were associated with food from east-ern European countries with a higher botulism inci-dence than that of the United Kingdom. In comparison, previously there were only four United Kingdom out-breaks between 1956 and 2001 (McLauchlin et al., 2006). There is also the need to be alert to the poten-tial transfer of botulinum neurotoxin genes to other bacteria. A more recent concern is the deliberate

spectrometer: detection and differentiation of the endo proteinase activities of botulinum neurotoxins A-G by mass spectrometry.

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