List Labs has been making botulinum toxins for research for 30 years.

In that time, we’ve made reagent and GMP products for researchers and pharma companies. Our products are of the highest quality and purity; they are produced in consistent processes, tested and stabilized by freeze-drying. Proteins that need to be activated by proteolytic cleavage (nicking) are activated and purified, providing consistent proteins for your research. You do not loose toxin in the nicking process, nor do you run experiments with toxin having a variable amount of nicking. We offer both pure neurotoxin and toxin complex, fully activated.

For work with the enzymatic light chains, we offer recombinant Light Chains in four different serotypes, LC/A, /B, /C and /D which are non-toxic and may be applied to research using common laboratory practices. Recombinant heavy chain, binding domains, both GST tagged and un-tagged are available. Our toxoids are made from purified neurotoxin types A and B to give you the most specific antibody production.

We have demonstrated the use of our type A toxin antibodies; one polyclonal raised against the heavy chain is an effective capture antibody for ELISAs and other detection strategies and the other antibody, a mouse monoclonal will specifically bind to type A light chain. This antibody pair will capture and identify small amounts of toxin.

The table below lists the Product #’s for these research reagents; several are offered in different sizes.

TOXINTYPE	COMPECE	NEUROTOXIN	TOXOID	CHAINS	ANTIBODIES
A	128	130	133	611, 612, 613	730, 731
B	138	138	139	620, 622, 623
C				625
D		146		630
E	140	141

If you have questions, please contact us at sales@listlabs.com for more information.

If you are an existing customer, you can place your order with a purchase order at orders@listlabs.com

If you are not yet a customer, fill out the customer app, once approved, we can fill your order.

See information outlining the purchase of controlled toxins on our website.

Orders of Select Agent products must total less than 1mg. There are no such limitations on antibodies or chains.

By: Hans Bigalke

Tetanus toxin (TeNT) and botulinum toxin (BoNT) appear quite different at first glance, however, when we take a closer look at how these toxins function, they are more similar than suggested by the diseases they cause.

Symptoms of Tetanus vs. Symptoms of Botulism

While tetanus causes a body to take a rigid, inflexible state, a very well-described and feared disease since antiquity; botulism reveals itself in limp, uncontrolled muscles, symptoms that mimic those of other diseases, hiding the cause of the disease until the modern age.

Symptoms are strikingly opposite: tetanus is characterized by unrelieved tension or spasticity of the striated muscles and botulism by a limp or flaccid state of the same muscles. In both cases, the muscles can no longer be moved in a coordinated manner, resulting in respiratory paralysis and death.

Tetanus and Botulism Have Similar Basic Origins and Structures

Both diseases can be attributed to toxins created by Clostridia. In general, TeNT is formed by bacteria introduced through injuries such as puncture wounds, placing the bacteria where they can grow in the absence of oxygen. BoNT is also synthesized by bacteria growing under lack of oxygen; however, in contrast to TeNT, botulinum is usually encountered when bacteria multiply and produce toxin in contaminated foods and the toxin is swallowed with the contaminated food. Both toxins enter the bloodstream and are distributed through the body (¹, ², ³, ⁴).

Botulinum and tetanus neurotoxins are both large proteins composed of two parts, a heavy chain, and a light chain. The light chain represents the active component; it is a protease that cleaves peptides regulating exocytosis of neurotransmitters, rendering the nerve unable to communicate. The heavy chain navigates the toxin into target cells and is responsible for transfers through several membranes.

Although botulinum and tetanus toxins have the same basic structure, tetanus neurotoxin exists solely as a two-part protein neurotoxin; where botulinum toxin is, at least initially, associated with accessory proteins, forming a toxin complex. This complex can be more than four times larger than the neurotoxin alone (⁵, ⁶).

TeNT is taken up by peripheral cholinergic nerve endings and is transported intraaxonally, retrogradely into the soma of the nerve cell (⁷). It leaves the motor neuron and subsequently enters nerve endings of inhibitory interneurons (⁸, ⁹, ¹⁰, ¹¹). Within the inhibitory neurons, the tetanus enzyme cleaves vesicular VAMP2, inhibiting the release of the transmitters glycine and GABA (¹²). With this action, the fine adjustment of the coordination of motor motion is disturbed. Inhibition is no longer possible so excitatory input is passed unfiltered from the spinal cord to the periphery. Minute peripheral sensory stimuli release a pronounced spasm, the clinical indication of tetanus. A similar muscle spasm is caused by strychnine, a blocker of glycine receptors.

In addition to this central effect, TeNT also has peripheral effects, splitting VAMP-2 in cholinergic nerve endings, leading to flaccid paralysis. However, this effect is triggered only at about 100-1000 fold higher concentrations, amounts of toxin which are not naturally encountered, so that peripheral effects play no role in clinical tetanus. Peripheral effects can be studied experimentally on isolated nerve-muscle preparations.

It turns out that TeNT largely mimics the effect of BoNTs (¹³). Both TeNT and BoNTs cleave vesicular proteins that trigger fusion of the transmitter-containing vesicles with the plasma membrane. Concentrations of BoNT needed to create paralysis are in general as low as the concentration of TeNT leading to the central effect. The BoNT serotype B not only splits the same protein as TeNT, it even cleaves it in the same place (¹⁴). Clearly, the difference between the action of botulinum and tetanus toxins is the location where the light chain is released and destroys the vesicle docking mechanism. Transport to the different sites of action is carried out by the heavy chains of these toxins. Surprisingly, BoNT/A and E also enter the soma of motor neurons by retrograde transport and eventually interneurons, where they can trigger central effects (¹⁵, ¹⁶, ¹⁷, ¹⁸, ¹⁹). These effects occur only at high concentrations and are masked by the peripheral paralysis.

Synapses must be actively sending or receiving neurotransmitters to allow endocytosis of both BoNT and TeNT. The reason for this lies in the localization of the receptors for these toxins on the luminal side of the synaptic vesicle. Only after the synaptic vesicle merges and becomes incorporated into the cell membrane do the receptors become accessible to the toxins. However, the dependence of uptake on synaptic activity is only valid if the peripheral effects are involved. Systemic TeNT, which is transported axonally, enters neurons by a different mechanism; it is endocytosed independent of synaptic activity (10, ²⁰). TeNT enters vesicles which transport peripheral metabolites via the retrograde route into the soma, for reuse or introduction into other metabolic pathways. TeNT travels on this route as a stowaway.

BoNT serotypes and TeNT are believed to be derived from an ancient toxin that has adapted to different targets in the course of evolution. An adaption allowing the toxin to readily reach a different destination in the nervous system is probably responsible for disguising the toxin.

TeNT FAQs

How similar are the amino acid sequences of TeNT and BoNT?
Why is TeNT not absorbed orally?
Can BoNT form in poorly perfused human tissue, similar to TeNT?
Is TeNT like BoNT also synthesized outside of a living organism, for example, in food?
BoNT is used therapeutically to treat pathological muscle cramping and spasticity. Are there any indications for TeNT, e.g. local paralysis after spinal injuries or stroke?
Can TeNT like other bacterial toxins be used as a tool in research?
Is the receptor known for TeNT?
Is the disease tetanus still a health problem?

Question: How similar are the amino acid sequences of TeNT and BoNT?

Answer: The similarity is about 40-50%, depending on the BoNT serotype.

Question: Why is TeNT not absorbed orally?

Answer: A complex of several proteins protects BoNTs from proteolytic degradation in the upper small intestine, this complex is responsible for the oral availability of botulinum toxins. During its further passage in the digestive system, as soon as the pH changes from acid to basic, neurotoxin leaves the complex and is able to enter the circulatory system. SinceTeNT has no protective complex proteins, like all other proteins, it is destroyed in the course of the gastrointestinal passage.

Question: Can BoNT form in poorly perfused human tissue, similar to TeNT?

Answer: Clostridium botulinum also grows in poorly perfused tissue injury and can form and release BoNT. Recently, such intoxications have been observed in drug addicts who use injectables contaminated with clostridial spores (²¹). BoNT is also formed in the intestine of infants when they consume spore-contaminated food, like honey.

Question: Is TeNT like BoNT also synthesized outside of a living organism, for example, in food?

Answer: At least under laboratory conditions, TeNT is produced from bacteria in fermenters. Whether Clostridium tetani naturally produce the toxin under oxygen deficiency outside living organisms is a good question. Tetanus toxin, without a protective coating, is more vulnerable to the environment than botulinum toxin complex. It does not survive the digestive process when ingested. Toxin produced outside of a living organism will likely not survive and would not provide a competitive advantage. From the point of view of the organism which uses toxin to secure food and a livable environment, making toxin which is destroyed would be a waste of energy.

Question: BoNT is used therapeutically to treat pathological muscle cramping and spasticity. Are there any indications for TeNT, e.g. local paralysis after spinal injuries or stroke?

Answer: Theoretically one could imagine such applications. In the developed world, however, the population is fully immunized against TeNT, so that injected toxin is immediately neutralized by specific antibodies. A similar situation occurs when antibodies are formed during therapy with BoNT and the BoNT becomes ineffective.

Question: Can TeNT like other bacterial toxins be used as a tool in research?

Answer: Several opportunities are offered by tetanus for research. TeNT serves as an aid to the study of axonal transport and has the potential to be used as a carrier for other proteins or substances that are to be channeled into the spinal cord. TeNT binds exclusively to neurons and as a result, is an excellent neuronal marker. For this purpose, either the toxin itself or the binding C-fragment can be equipped with a tag like FITC or detected by standard immunology. Finally, tetanus toxoid is an excellent carrier for antigens used to develop vaccines (²²).

Question: Is the receptor known for TeNT?

Answer: The receptor tetanus toxin is unknown. However, the toxin has two pockets in the binding domain that could recognize different receptors. It is suggested that the receptor responsible for peripheral paralysis is located on the inside of synaptic vesicles like the receptors for the other clostridial neurotoxins and that the receptor that transports the toxin axonally is accessible to the toxin independently of exocytosis. TeNT like BoNT/A is bound to polysialo-gangliosides that reside on the outer side of the plasma membrane of neurons.

Question: Is the disease tetanus still a health problem?

Answer: With the help of immunization of the population against the disease, tetanus occurs only rarely and in unimmunized people. The WHO recommends boost injections every ten years. Tetanus is quite a problem in developing countries. In some states in Africa for example, many infants die from Clostridium tetani infections that occur when umbilical cords are cut with contaminated tools.

References:

Popoff MR, Bouvet P 2009 Oct; “Clostridial toxins“ Future Microbiol. 4(8):1021-64. PMID: 19824793

Rossetto O, Pirazzini M, Bolognese P, Rigoni M, Montecucco C. 2011 Dec; “An update on the mechanism of action of tetanus and botulinum neurotoxins” Acta Chim Slov. 58(4):702-7 PMID: 24061118

Binz T, Rummel A. 2009 Jun; “Cell entry strategy of clostridial neurotoxins” J Neurochem. 109(6):1584-95. PMID: 19457120

Pirazzini M, Rossetto O, Eleopra R, Montecucco C 2017 Apr, “Botulinum Neurotoxins: Biology, Pharmacology, and Toxicology“ Pharmacol Rev 69(2):200-235 PMID: 28356439

Gu S, Rumpel S, Zhou J, Strotmeier J, Bigalke H, Perry K, Shoemaker CB, Rummel A, Jin 2012 Feb “Botulinum neurotoxin is shielded by NTNHA in an interlocked complex” 24;335(6071):977-81. PMID: 22363010

Benefield DA, Dessain SK, Shine N, Ohi MD, Lacy DB 2013 Apr; “Molecular assembly of botulinum neurotoxin progenitor complexes“ Proc Natl Acad Sci USA 110(14):5630-5 PMID: 23509303

Erdmann G, Wiegand H, Wellhöner HH. 1975 “Intraaxonal and extraaxonal transport of 125I-tet- anus toxin in early local tetanus” Naunyn Schmiedebergs Arch Pharmacol. 290(4):357- 73 PMID: 53793

Surana S, Tosolini AP, Meyer IFG, Fellows AD, Novoselov SS, Schiavo G. 2018 Jun “The travel diaries of tetanus and botulinum neurotoxins” Toxicon 1;147:58-67. PMID: 29031941

Bercsenyi K, Giribaldi F, Schiavo G. 2013; “The elusive compass of clostridial neurotoxins: deciding when and where to go?” Curr Top Microbiol Immunol. 364:91-113 PMID: 23239350

Lalli G, Bohnert S, Deinhardt K, Verastegui C, Schiavo G. 2003 Sep; “The journey of tetanus and botulinum neurotoxins in neurons” Trends Microbiol. 11(9):431-7. PMID: 13678859

Schwab ME, Thoenen H. 1976 Mar “Electron microscopic evidence for a transsynaptic migration of tetanus toxin in spinal cord motoneurons: an autoradiographic and morphometric study” Brain Res. 26;105(2):213-27 PMID: 1260442

Brunger AT, Rummel A. 2009 Oct; “Receptor and substrate interactions of clostridial neurotoxins” Toxicon. 54(5):550-6 PMID: 19268493

Schmitt A, Dreyer F, John C. 1981; “At least three sequential steps are involved in the tetanus toxin-induced block of neuromuscular transmission” Naunyn Schmiedebergs Arch Pharmacol. 317(4):326-30. PMID: 6119629

Schiavo G, Benfenati F, Poulain B, Rossetto O, Polverino de Laureto P, DasGupta BR, Montecucco C. 1992 Oct “Tetanus and botulinum-B neurotoxins block neurotransmitter release by pro- teolytic cleavage of synaptobrevin” Nature. 29;359(6398):832-5. PMID: 1331807

Wiegand H, Erdmann G, Wellhöner HH. 1976; “125I-labelled botulinum A neurotoxin: pharmaco- kinetics in cats after intramuscular injection” Naunyn Schmiedebergs Arch Pharmacol. 292(2):161-5 218

Restani L, Giribaldi F, Manich M, Bercsenyi K, Menendez G, Rossetto O, Caleo M, Schiavo G. 2012 Dec; “Botulinum neurotoxins A and E undergo retrograde axonal transport in primary motor neurons” PLoS Pathog. 8(12) PMID: 23300443

Wiegand H, Wellhöner HH. 1977 Jul; “The action of botulinum A neurotoxin on the inhibition by antidromic stimulation of the lumbar monosynaptic reflex” Naunyn Schmiedebergs Arch Pharmacol. 298(3):235-8. PMID: 895899

Restani L, Novelli E, Bottari D, Leone P, Barone I, Galli-Resta L, Strettoi E, Caleo M. 2012 Aug; “Botulinum neurotoxin A impairs neurotransmission following retrograde transynaptic transport” 13(8):1083-9. PMID: 22519601

Caleo M, Restani L. 2018 Jun “Direct central nervous system effects of botulinum neurotoxin” Toxicon. 1;147:68-72 PMID: 29111119

Bohnert S, Schiavo G. 2005 Dec “Tetanus toxin is transported in a novel neuronal compartment characterized by a specialized pH regulation” J Biol Chem. 23;280(51):42336-44. PMID: 16236708

Gonzales y Tucker RD, Frazee B. 2014 Dec; “View from the front lines: an emergency medicine perspective on clostridial infections in injection drug users” Anaerobe. 30:108-15 PMID: 25230330

Aba YT, Cissé L, Abalé AK, Diakité I, Koné D, Kadiané J, Diallo Z, Kra O, Oulaï S, Bissagnéné E. 2016 Aug; “[Neonatal and child tetanus morbidity and mortality in the University hospitals of Abidjan, Côte d’Ivoire (2001-2010)]” Bull Soc Pathol Exot. 109(3):172-9 PMID: 27177642

By: T.J. Smith

The answer as to whether the botulinum neurotoxin (BoNT)-producing bacteria comprised six separate species required a complete revolution in microbial classification. Up to the turn of the last century, bacterial differentiations were based on morphological characteristic and biochemical activities, known collectively as phenotypic characteristics. However, the discovery of DNA as the ultimate code of life led to technological methodologies enabling the sequencing and comparisons of individual genes and, ultimately, entire bacterial genomes. Initial studies used DNA-DNA hybridization (DDH) techniques which, due to the cumbersome nature of the assays, was followed quickly by comparative analyses of 16s rRNA gene, which is a highly conserved gene that is present among all bacterial species (Rossello-Mora and Amann 2001). The results of these studies were remarkably similar, providing confidence in the predictability of both assays for bacterial speciation.

Setting the Stage for Current Classifications

16s rRNA analyses of various clostridial species verified earlier thoughts about their relationships (Collins 1998). The proteolytic BoNT type A, B, and F-producing C. botulinum bacteria were found to cluster with closely related C. sporogenes, while nonproteolytic BoNT type B, E, and F-producing C. botulinum were determined to be a distinct species cluster. Type C and D-producing bacteria were closely related to non-neurotoxigenic C. novyi strains. Type G-producing bacteria, along with nontoxic C. subterminale, were deemed a distinct species, designated C. argentinense. Type F-producing C. baratii and type E-producing C. butryricum were both found to be indistinguishable from their nontoxic counterparts using these techniques. Thus, in addition to the neurotoxin-producing bacteria that had reverted to nontoxicity, additional connections between toxic and nontoxic organisms were seen. This completely contradicted the theory that any botulinum neurotoxin-producing bacteria should be named “C. botulinum” and set the stage for current classifications based on whole genome analysis for differentiation of bacterial strains.

Seven Distinct Clostridial Species Produce Botulinum Neurotoxins

Currently, this analysis can be done at a very fine level, as each of the approximately 4 million nucleotide residues that reside within an average clostridial genome can be identified and compared. Individual nucleotide differences among core, or shared, genes within a genome are analyzed using numerical computations that help determine species/species interfaces (Richter and Rossello-Mora 2009). This technique is known as average nucleotide identity, or ANI. It is known that the same bacterial isolate can mutate over time in the laboratory, so that sequencing of the same isolate over time should show a few minimal differences. However, larger scale differences are seen in different strains within the same species and further numbers of differences separate distinct species. These relationships are strengthened through analysis of large numbers of genomes, and this has helped to support an avalanche of bacterial genome sequencing studies. To date over 200 Clostridium botulinum strains plus over 60 closely related strains have been sequenced and subjected to comparative analysis (https://www.ncbi.nlm.nih.gov/pubmed). The results confirm that seven distinct clostridial species are capable of producing botulinum neurotoxins (Williamson, Sahl et al 2016). These include three groups and four species. The first, Group I, proteolytic C. botulinum, had a name change, to C. parabotulinum and then changed back to C. botulinum Group I (Smith, Williamson et al 2018); Group II includes the nonproteolytic C. botulinum type B, E, and F toxin producers, and Group III, type C and D toxin-producing C. botulinum, a group name which has had a suggested change to C. novyi sensu lato (Skarin, Hafstrom et al 2011). In addition to these groups, four genetically distinct species which may produce botulinum toxin are C. argentinense; C. baratii; C. butyricum; and C. sporogenes.

Different species may produce the same toxin and different toxins may be produced by the same bacterial species. In addition, there are documented non-neurotoxigenic members represented in each species. A listing of BoNT-producing bacteria and their characteristics is shown in Table 1 (Hatheway 1988, Collins 1998).

Table 1. An abbreviated table showing some major characteristics of various clostridia, that produce botulinum toxin.

Species/group	Toxins produced	Lipase	Lecithinase	Proteolytic
C. botulinum Group I	A, B, F, Ab, Ba, Af, HA	+	–	yes
C. botulinum Group II	B, E, F	+	–	no
C. botulinum Group III	C, D	+	variable	variable
C. argentinense	G	–	–	yes
C.baratii	F	–	+	no
C. butyricum	E	–	–	no
C. sporogenes	B	+	–	yes

It has been determined that there is a great deal of diversity among the bacteria that produce botulinum toxins, as well as among the toxins themselves. The seven toxin serotypes differ to such a large extent that the antisera to one type cannot neutralize the toxin of a different type. However, genetic analysis of these toxins has revealed yet another level of diversity. The identification and study of BoNT subtypes has been the subject of increasing interest in the past three decades, leading to a whole new understanding of the complexity of these proteins.

About List Labs

List Labs offers over 100 reagents including Botulinum Toxins. These products are used in a wide variety of scientific research studies. You can read about some of them on our citation page. Contact us today to discuss your next project.

About the Author

Theresa Smith has studied botulinum neurotoxins for over 25 years, specializing in toxin countermeasure research, and is considered a leading expert regarding diversity in botulinum neurotoxins as well as the organisms that produce these toxins.

References

Collins, M. D. (1998). “Phylogeny and taxonomy of the food-borne pathogen Clostridium botulinum and its neurotoxins.” J Appl Microbiol 84: 5-17. PMID: 15244052

Hatheway, C. L. (1988). Botulism. In A. Balows, W. H. Hausler, J. Ohashi and A. Turano (ed) Laboratory Diagnosis of Infectious Diseases New York, Springer-Verlag: 111-133.

Richter, M. and R. Rossello-Mora (2009). “Shifting the genomic gold standard for the prokaryotic species definition.” Proc Natl Acad Sci U S A 106(45): 19126-19131. PMID: 19855009

Rossello-Mora, R. and R. Amann (2001). “The species concept for prokaryotes.” FEMS Microbiol Rev 25(1): 39-67. PMID: 11152940

Skarin, H., T. Hafstrom, J. Westerberg and B. Segerman (2011). “Clostridium botulinum group III: a group with dual identity shaped by plasmids, phages and mobile elements.” BMC Genomics 12(185): 1-13. PMID: 21486474

Smith, T. J., C. H. Williamson, K. Hill, J. W. Sahl and P. Keim (2018). “Botulinum neurotoxin-producing bacteria – isn’t it time we called a species a species?” MBio in press.

Williamson, C. H., J. W. Sahl, T. J. Smith, G. Xie, B. T. Foley, L. A. Smith, R. A. Fernandez, M. Lindstrom, H. Korkeala, P. Keim, J. Foster and K. Hill (2016). “Comparative genomic analyses reveal broad diversity in botulinum-toxin-producing Clostridia.” BMC Genomics 17: 180. PMID: 26939550

By: T.J. Smith

Gram strain of C. botulinum

Origins of Botulinum Toxin Types – relationships between toxins and the bacteria that produce them

Soon after the discovery that botulism was caused by a toxin, multiple toxin types were identified. Initial characterizations were based mainly on serological differences, however other anomalies were noted, such as differences in toxicity, sensitivity or resistance of different animal species to intoxication, cultural or morphological characteristics, and, finally, genetic differences.

Historical Differentiation of Bacterial Organisms

In the late 1800s and early 1900s, differentiation of bacterial organisms was mostly a matter of observations related to colony and cell characteristics, growth characteristics, and biochemical usage. Organisms were often partly identified according to their Gram stain characteristics, either Gram stain positive (purple) or Gram negative (red), often with cocci (balls) or rod (rectangular) shapes. Variations in shape, size, or coloration served to further delineate certain genera, such as with the paired kidney bean shapes of Neisseria or the comma-shaped Vibrios. The presence of Gram positive organisms with subterminal oval spores served to further identify the anaerobic bacteria as Clostridium.

Additional delineations have come from biochemical reactions of the bacteria. These may involve the ability to break down and utilize different proteins in the environment (proteolysis) or to utilize various carbohydrates through sugar fermentation. Examples of these assessments include liquification of gelatin or color changes triggered by a lowered pH due to fermentation of lactose or sucrose. With toxin-producing clostridial species, the ability to break down fats using lipases (positive lipase reaction) coupled with an inability to break down lecithin (negative lecithinase reaction) were hallmarks of the presence of Clostridium botulinum. Differences in optimal growth temperatures and resistance of spores to heat treatment have also been used as tools for differentiation.

Different Bacterial Variants Found to Produce Both Same and Different Toxins

Differences in these characteristics were noted from the beginning, when the Ellezelles strain characterized by Dr. E. Van Ermengem was found to be a nonproteolytic organism that favored a moderate optimal growth temperature of 25-30° C, while the Darmstadt strain identified by Dr. G. Landmann was definitely proteolytic with a higher optimal growth temperature of approximately 37° C (Van Ermengem 1897, Leuchs 1910). The Darmstadt strain produced type A toxin, while the Ellezelles strain produced type B toxin. The fact that the two strains produced different toxin serotypes initially linked these toxin differences with the bacterial differences. However, it was quickly discovered that different bacterial variants could produce the same toxin and the same bacteria could produce different toxins. C. botulinum strains that produced type A toxin were identified from the west coast of the United States, while virtually identical strains from the east coast were identified as type B (Burke 1919). However, the bacteria producing type B toxins in Europe differed from those in the U.S. in that the European strains were nonproteolytic, while the U.S. strains were uniformly proteolytic. This provided clear evidence that the bacterial types and the toxins they produced were not linked.

In 1922, the literature began to reflect these bacterial differences by identifying proteolytic organisms that produce botulinum neurotoxin as “Clostridium parabotulinum” and nonproteolytic organisms remained “Clostridium botulinum”. Dr. H. R. Seddon first used the term C. parabotulinum to distinguish his type C strains, isolated from cattle in Australia, from those of Dr. Ida Bengtson, isolated from fly larvae in the U.S. (Bengtson 1922, Seddon 1922). In addition to difficulties encountered when neutralizing her toxins with his antisera, the strains themselves appeared to differ in proteolytic tendencies. For the next 30 years proteolytic type A and B strains and Seddon type C strains were labeled C. parabotulinum while the nonproteolytic European type B strains and U.S. type C strains were designated C. botulinum.

When type E-producing bacteria were characterized, they were found to be uniformly closely related to the nonproteolytic type B strains (Hazen 1937), and on rare occasions both proteolytic and nonproteolytic bacterial strains that produced type F toxin were isolated (Moller and Scheibel 1960, Eklund, Poysky et al. 1967).

Bacterial Variants of Botulinum Toxins

Despite these obvious bacterial strain differences, it was proposed in 1953 and decided over the following decade to designate all botulinum neurotoxin-producing organisms as “Clostridium botulinum” on the basis of that single overriding characteristic. This was problematic as bacterial strains were already known that had produced botulinum toxin in the past but were no longer toxin producers. A major surprise came with the identification of an entirely different clostridial species, C. baratii, that produced type F toxin (Hall, McCroskey et al. 1985). Shortly after this came the identification of a C. butyricum strain that produced type E toxin (Aureli, Fenicia et al. 1986). In addition, the characterization of the bacteria that produced type G toxin revealed that it was a distinct species, prompting its designation as C. argentinense (Gimenez and Ciccarelli 1970).

Based on phenotypic characteristics, at least six different bacterial variants that could produce one (or more) botulinum neurotoxins have been identified. The question of whether these variants are a single entity or represent separate species was later answered using technological advances in genetic analyses.

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About the Author

References

Aureli, P., L. Fenicia, B. Pasolini, M. Gianfranceschi, L. M. McCroskey and C. L. Hatheway (1986). “Two cases of type E infant botulism caused by neurotoxigenic Clostridium butyricum in Italy.” J Infect Dis 154(2): 207-211.

Bengtson, I. (1922). “Preliminary note on a toxin-producing anaerobe isolated from the larvae of Lucilia caesar.” Pub Health Repts 37: 164-170.

Burke, G. S. (1919). “Notes on Bacillus botulinus.” J Bact 4: 555-571.

Eklund, M. W., F. T. Poysky and D. I. Wieler (1967). “Characteristics of Clostridium botulinum type F isolated from the Pacific Coast of the United States.” Appl Microbiol 15(6): 1316-1323.

Gimenez, D. F. and A. S. Ciccarelli (1970). “Another type of Clostridium botulinum.” Zentralbl Bakteriol Parasitenk Infektionskr Hyg Abt 215: 221-224.

Hall, J. D., L. M. McCroskey, B. J. Pincomb and C. L. Hatheway (1985). “Isolation of an organism resembling Clostridium barati which produces type F botulinal toxin from an infant with botulism.” J Clin Microbiol 21(4): 654-655.

Hazen, E. L. (1937). “A strain of B. botulinus not classified as type A, B, or C.” J Infect Dis 60: 260-264.

Leuchs, J. (1910). “Beitraege zur kenntnis des toxins und antitoxins des Bacillus botulinus.” Z Hyg Infekt 76: 55-84.

Moller, V. and I. Scheibel (1960). “Preliminary report of an apparently new type of Cl. botulinum ” Acta Path Microbiol Scand 48: 80.

Seddon, H. R. (1922). “Bulbar paralysis in cattle due to the action of a toxicogenic bacillus, with a discussion on the relationship of the condition to forage poisoning (botulism).” J Comp Path Ther 35: 147-190.

Van Ermengem, E. (1897). “A new anaerobic bacillus and its relation to botulism (originally published as “Ueber einen neuen anaeroben Bacillus und seine beziehungen zum botulismus” in Zeitschrift fur Hygiene und Infektionskrankheiten, 26:1-56) ” Clin Infect Dis 4: 701-719.

3D Rendering of Botulinum Toxin Protein

By: T.J. Smith

Following a botulism outbreak due to contaminated ham that severely sickened 10 and resulted in the death of three people in Ellezelles, Belgium, a review and case study on botulism was published by a researcher named Emile Van Ermengem (Van Ermengem 1897). While Van Ermengem was not the first to study this syndrome, his article supplied critical information defining botulism as a type of food poisoning having specific paralytic symptoms. He determined that the illness was an intoxication, not an infection, and that its cause was a bacterial toxin. He was also able to isolate and characterize the organism responsible for the toxin as an anaerobic spore-forming bacillus, which he named Bacillus botulinus (later renamed Clostridium botulinum). “Botulinus” is the latin word for sausages, and this nomenclature was used due to the historic link between botulism and improperly processed sausages, particularly blood sausages. His careful and painstaking research provided the foundation for future studies on botulism, its causes and treatments.

During this time, botulism was thought to be related specifically to improperly processed meat, such as sausages and ham, and caused by a single monospecific toxin. Within the next decade both of these theories were proven wrong. In 1904, Dr. G. Landmann isolated a botulinum toxin-producing bacterial strain from preserved bean salad which had caused 11 deaths in Darmstadt, Germany (Landmann 1904). This was the first reporting of botulism due to a source other than meat or fish. In 1910, Leuchs showed that the Ellezelles strain from Van Ermengem and the Darmstadt strain from Landmann were immunologically distinct toxins, providing the first evidence that all botulinum neurotoxins were not the same (Leuchs 1910). This marked the beginning of a century of study related to botulinum neurotoxin diversity which included clinical case reviews and morphological, immunological, and, most recently, genetic studies.

As the bacteria responsible for botulinum neurotoxins initially seemed to be nearly identical in morphology and cultural characteristics, early delineations were the result of serological studies, which were apparently the “hot new thing” of the day. Antisera produced using a particular bacterial strain was tested against other strains to determine relationships among both the toxins and the bacteria that produced them. The assays were both qualitative and quantitative and included agglutination, immuno-absorption, and neutralization techniques (Schoenholz and Meyer 1925). They were originally targeted to both the toxins and the bacteria that produced them, but the emphasis quickly shifted to neutralization of toxins using specific antisera. These antisera, which were predominantly of equine origin, were developed for treatment purposes as well, and they continue to be the only approved treatment for botulism to this day. In 1919, Georgina Burke produced antisera from three strains isolated in California, Oregon, and New York, and she was able to show that the toxins from the two West Coast strains were immunologically identical, while the New York toxin was distinct (Burke 1919). She identified these toxins as type A (West) and Type B (East). Later studies of U. S. strains by K. F. Meyer and B. Dubovsky substantiated her findings (Meyer and Dubovsky 1922).

In the following decades several additional serotypes were identified. In 1922, Dr. Ida Bengtson reported a toxin from a C. botulinum strain that was not neutralized by either type A or type B antisera, which she designated type C (Bengtson 1922). The bacterial strain was isolated from fly larvae that proved to be causative agents in the intoxication of chickens that had ingested these larvae. This illustrates that botulism is not restricted to humans but rather can be seen in a wide variety of animals as well. In fact, differential sensitivities of the toxins in animals has formed a background for discerning various toxin types. In addition, catastrophic losses due to botulism have been noted in domestic fowl, cattle, horses, and even minks and foxes, prompting the development and use of vaccines in these animals for protection. H. R. Seddon isolated a culture that apparently produced type C toxin from an outbreak in cattle in Australia (Seddon 1922). The toxin could be neutralized by Bengtson’s antisera, however, the reverse was not true. This “one-way” neutralization was the first of several anomalies that were discovered when serotyping botulinum neurotoxins.

In 1929, Meyer and Gunnison showed that the toxin from a culture isolated by Theiler and associates in South Africa was immunologically distinct from types A, B, or C. This toxin, which was also related to intoxication in cattle, was designated type D (Meyer and Gunnison 1929).

In the following decade, several botulism cases were noted that were related to ingestion of fish. While Russian scientists were the first to note these unusual cases of botulism, it was Dr. Janet Gunnison who determined the toxins were a new type, and Dr. Elizabeth Hazen who published initial reports on type E botulism cases (Gunnison, Cummings et al. 1936, Hazen 1937). Outbreaks due to dried, smoked, or fermented fish, fish eggs, whale blubber, and seal or walrus meat are common, but there have been rare type E cases related to other foods as well.

The first case due to type F was linked to an outbreak involving duck paste on Langeland Island, Denmark, in 1958 (Moller and Scheibel 1960). Reported cases due to type F are rare and have been restricted to humans so far. Type G was isolated from a cornfield in Argentina in 1969 as part of a soil sampling study conducted by Dr D. F. Gimenez and Dr. A. S. Ciccarelli (Gimenez and Ciccarelli 1970). This type is unusual in that there are no direct reports of intoxications due to type G in people or animals. However, a study of autopsy materials related to sudden deaths due to unknown causes in Switzerland identified type G producing organisms among the samples (Sonnabend, Sonnabend et al. 1981). Why type G is only found in Argentina or Switzerland is a mystery.

As of 1970, there were seven known immunologically distinct botulinum toxin types. However, as we will discover, this was just the beginning of our understanding of the diversity that is seen within botulinum neurotoxins.

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References

Bengtson, I. (1922). “Preliminary note on a toxin-producing anaerobe isolated from the larvae of Lucilia caesar.” Pub Health Repts 37: 164-170.

Burke, G. S. (1919). “Notes on Bacillus botulinus.” J Bact 4: 555-571.

Gimenez, D. F. and A. S. Ciccarelli (1970). “Another type of Clostridium botulinum.” Zentralbl Bakteriol Parasitenk Infektionskr Hyg Abt 215: 221-224.

Gunnison, J. B., et al. (1936). “Clostridium botulinum type E.” Proc Soc Exp Biol Med 35: 278-280.

Hazen, E. L. (1937). “A strain of B. botulinus not classified as type A, B, or C.” J Infect Dis 60: 260-264.

Landmann, G. (1904). “Uber die ursache der Darmstadter bohnenvergiftung.” Hyg Rundschau 10: 449-452.

Leuchs, J. (1910). “Beitraege zur kenntnis des toxins und antitoxins des Bacillus botulinus.” Z Hyg Infekt 76: 55-84.

Meyer, K. F. and B. Dubovsky (1922). “The distribution of the spores of B. botulinus in the United States. IV.” J Infect Dis 31: 559-594.

Meyer, K. F. and J. B. Gunnison (1929). “South African cultures of Clostridium botulinum and parabotulinum. XXXVII with a description of Cl. botulinum type D, N. SP.” J Infect Dis 45: 106-118.

Moller, V. and I. Scheibel (1960). “Preliminary report of an apparently new type of Cl. botulinum ” Acta Path Microbiol Scand 48: 80.

Schoenholz, P. and K. F. Meyer (1925). “The serologic classification of B. botulinus.” J Immunol 10: 1-53.

Sonnabend, O., et al. (1981). “Isolation of Clostridium botulinum type G and identification of type G botulinal toxin in humans: report of five sudden unexpected deaths.” J Infect Dis 143: 22-27.

By: Mary N. Wessling, Ph.D. ELS

List Biological Laboratories’ (List Labs) catalog of products is related to furthering research in human health and preventing disease, most commonly as the starting materials for vaccine research & development or production around the world. Vaccines are mainly identified for their capacity to prevent diseases that the body’s innate defensive mechanisms (the skin and specialized cells in the blood, for example) can’t resist unaided. However, there are many other uses for these purified materials in medical research, and you will likely encounter wording on our website that is not part of everyday vocabulary for non-scientists. This article is intended to provide a basic understanding of some of the more frequently used terms and aid you in selecting the products most essential to your projects.

Toxin vs. Toxoid

For starters, what is the difference between “toxin” and “toxoid”. Broadly defined, anything that can cause harm to an organism is a toxin. However, for List Labs’ products and in biological usage, a toxin refers to a bacterial or viral product that has harmful effects when it enters the body (List Labs’ toxins are in a highly purified form). A toxoid is a chemically altered toxin that has reduced or no toxicity and is used for its remaining antigenic activity, which can stimulate an immune response.

Take, for example, cholera, a disease produced by Vibrio cholerae bacteria, possibly through contact with body fluids from a person ill with cholera or through contaminated water supply. Cholera causes severe diarrhea, and untreated, it can be fatal. However, the purified List Labs’ cholera toxin by chemical modification becomes a toxoid that lacks toxic activity but retains structures that make it useful for immunization of research animals or stimulation of immune cells in vitro.

How do Toxoids Impact the Immune Process?

To understand how some List Labs products work, an overview of the immune process is helpful. During the course of a day, we frequently touch, ingest, or breathe in something that has potential to harm the body. Our cells react to this invader: is this a threat, or not, and if so, how serious is the threat?

What is the Innate Immune Response?

The innate immune response is the first order of defense in the immune process. There are many different cell types in our body. Some of these cells are equipped by their structural and biochemical components to destroy dangerous microbial invaders–pathogens–quickly. The inflammation that we experience from minor infections is often a sign of this process as cells from the blood destroy the pathogen. This happens quickly, within hours.

What is the Adaptive Immune Response?

Another cellular response system requires a longer time to react to the threat. These cells react by changing from an inactive form to one that will start a more complex defensive process: this is the second step, the adaptive immune response. There are two different classes of cells that comprise the adaptive immune response; they differ by the structures that give them their ability to bind antigens– the invading bacteria and viruses. Both these cells are called lymphocytes; individually, they are the B-lymphocytes (B-cells) and T-lymphocytes (T-cells). Both originate from stem cells in the bone marrow; B and T refer to the place in the body where they mature. T-cells mature in the thymus into several subclasses of T-cells that circulate in the blood and lymph. “Killer” T-cells recognize foreign antigens on cell surfaces (e.g. from viral infection or malignancy). “Helper” T-cells induce B-cells to produce antibodies. “Suppressor” T-cells dampen or regulate the immune response to prevent over-reaction. B-cells mature in the bone marrow and migrate to secondary lymphoid tissues (e.g. spleen and lymph nodes). When they encounter foreign antigens and/or helper T-cells, they are stimulated to divide and expand clonally to produce antibodies and differentiate into plasma cells.

What is Immune Memory?

After the B and T-lymphocytes react to an antigen, two results are possible. The first, and desirable result, is that the invader is identified and defeated, leaving behind what might be called its criminal record: immune memory. When the antigen comes creeping back in the future, the adaptive immune system recognizes it and attacks. The second possibility is an over-reaction and lack of cessation of the adaptive immune process that is harmful to the body: an autoimmune condition.

Antigens, Epitopes and Vaccines

Where do vaccines come into this process? An antigen is a substance that causes the body to mount an immune response against it. Antigens include toxins, bacteria, viruses, or other substances that the body recognizes as foreign or not “self”. Vaccines have structural features similar to structures of the toxin or invading pathogen that can elicit adaptive immunity.

An epitope is a specific molecular region on the surface of an antigen, typically one of many on the antigen, that elicits an immune response and is capable of binding with the specific antibody produced by the response. A toxin has many epitopes that can be recognized by the immune response. The epitopes that are required for toxicity have been altered chemically in toxoids or by specific genetic mutations in inactive mutants; however, many epitopes are retained and can stimulate an adaptive or memory immune response that will be effective against the toxin.

Toxins and Toxoid Products for Research

Below is a list of toxin and toxoid or inactive mutant pairs of products available to support your research.

Toxin and product numbers	Toxoid	Inactive Mutant
Botulinum Neurotoxin Type A from Clostridium botulinum – 130A, 130B, 9130A	133L
Botulinum Neurotoxin Type B, Nicked, from Clostridium botulinum – 136A, 136B	139
Toxin A from Clostridium difficile – 152C	153
Toxin B from Clostridium difficile – 155A, 155B, 155L	154A
Diphtheria Toxin, Unnicked, from Corynebacterium diphtheriae – 150	151	149
Enterotoxin Type B from Staphylococcus aureus – 122	123
Tetanus Toxin from Clostridium tetani – 190A, 190B	191A, 191B

By: Rachel Berlin, Marketing Manager

The List Labs website hosts a library of scientific article abstracts related to the research performed using our products called the Citations Page. Visitors can search this library to learn how others have used List Labs’ reagents in their research. This valuable resource is updated monthly with new articles from a wide variety of publications. Check out a few recent articles below:

Botulinum Neurotoxin

Carrier Proteins

Clostridium difficile toxin

Lipopolysaccharide (LPS)

Diphtheria toxin

Don’t see the reagent you’re interested in? You can search the citations by product, year, publication, or by the type of cell, animal, assay, protein or research. Check it out today!

List Biological Laboratories, Inc. (List) actively supports and participates in the BabyBIG^® project.

What is BabyBIG^®?

BabyBIG^® (Human Botulism Immune Globulin; BIG-IV) is a public service, not-for-profit orphan drug manufactured and distributed by the California Department of Public Health.It is the only therapy available for infants who are infected with the organism that causes botulism, a life-threatening disease.

List Labs Volunteers Donate Plasma to Support Orphan Drug BabyBIG^®

Because List produces the botulinum toxin for research use, employees are vaccinated against the toxin, thereby producing antibodies which circulate in their plasma. This puts List Laboratories in a rare position to help with this project. These antibodies are donated by volunteer employees via plasmapheresis, a procedure similar to a blood donation, for a period of up to 12 weeks. Life-saving plasma is blended and processed into the final BabyBIG^® product. We are proud of being able to be a big part of this amazing product and effort. There are only a handful of organizations and entities who would be able to participate at any level and over 1/3 of our employees are active donors. We salute them and support them in their time commitment to a worthy cause.

Infant Botulism Patients Helped in a Big Way by BabyBIG^®

Since licensure of BabyBIG^® in 2003, approximately 1100 infant botulism patients nationwide have been treated with it, thereby shortening each hospital stay by almost one month and reducing the negative impact of this disease on these young patients. In the aggregate since licensure, treatment with BabyBIG^® has resulted in more than 65 years of avoided hospital stay and more than $100 million in avoided hospital costs.

More information about BabyBIG^® may be found on their web site www.infantbotulism.org