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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.
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.
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.
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.
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.
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.
Debra Booth, VP of Operations
Linda Eaton, Ph.D., VP of Research & Development
Stacy Burns-Guydish, Ph.D., Senior Director of Microbiology
PJ Nehil, Sales & Distribution Coordinator
Dear Researchers Everywhere,
List Labs recently exhibited at the 2016 BIO International Convention. As a producer of bacterial products for research, as well as a provider of custom laboratory services, we were excited to meet with current and potential customers. We were eager to gain some insight into the current and future state of biotechnology and the up and coming field of microbiome research. But you never know exactly how you’ll feel until you spend the time in the conference exhibit hall. We were very pleased to see that we are a part of an industry that is moving forward at the pace of a start up, fueled by the novel ideas and intellect of many scientists.
Last week, thousands of people filled the Moscone Convention Center in San Francisco. The event was brilliantly organized and the venue was strategically arranged. The various pavilions were organized by state or country and some by specialty. It was a great opportunity to network and identify new sources for projects and services. Attendees were CEO and business development folks interested in learning more about what exhibitors have to provide. At our booth, we talked about immunotherapy, live biotherapeutics, contract manufacturing, GMP production, and more. It was a pleasure to shake hands with many customers, distributors, and colleagues and discuss ways we can partner with them to move their research forward.
We also had the opportunity to meet with vendors, in shipping, supplies and services. These vendors are critical to delivery of our biological and therapeutic products, and we benefited from learning about their new offerings as strategic partners. The enthusiasm was palpable from both exhibitors and attendees. At this event, we didn’t just meet industry veterans. We met many young scientists and job seekers looking for their first break. Some even came directly to us to hand-deliver their resumes. The event had a job expo, fueling another layer of energy and opportunity for exhibitors. We were resident in the California Pavilion where we learned that the State of California has a Biosciences Training Program, which will help companies pay for new employment training. Community colleges around the country are encouraging students to contribute to the future of biotechnology through clinical and regulatory apprenticeships. It’s great to see that science is providing opportunity for students in so many ways.
In closing, we found the 2016 BIO International Convention to be highly productive for our company. For those of you who didn’t get a chance to meet us at the convention, it’s very easy to reach us online and on social media. We would love to connect with you on LinkedIn, tweet with you on Twitter, like each other on Facebook and Google+. You can also check these accounts if you’re curious about your next opportunity to meet us at a conference. We even have a YouTube channel and a blog where you can learn more about us. We’d love to see your YouTube videos and read your blog if you have them as well. The future of biotechnology looks bright and we’re more excited than ever to be a part of it. We will definitely attend more events like this and we hope to see you at BIO 2017 in San Diego!
Debra, Linda, Stacy, & PJ
UPDATE: July 13, 2016
Alpha Toxin from C. septicum, product #116L is now available for purchase online. Information on other potential upcoming products can be found on our Product Pipeline.
UPDATE: May 16, 2016
Alpha Toxin from C. septicum is currently in late to final stages of development, and we are accepting orders now. We expect to ship our first orders toward the end of this month or the earlier part of June. Please e-mail your purchase order to ORDERS@listlabs.com.
Product details are as follows:
Documents for Product #116L (safety data sheet, certificate of analysis, etc.) will be posted on our website when available. Official availability from stock will be announced in a future update on this blog post. We will also make announcements on social media. Check our Facebook, Twitter, LinkedIn, and Google+, or contact us directly if you would like to be among the first to know when Product #116 is officially available.
Originally published on October 26, 2015
By: Md. Elias, Ph.D, Senior Scientist
List Labs is constantly bringing new products as research reagents, and GMP grades, to the scientific community for the advancement of science. A recombinant C. septicum alpha toxin will soon be added to our product list and can be found in our pipeline information. This toxin can be useful for basic research, immune assay development, vaccine development and more importantly in cancer research. If you have specific interest in this product, please contact us for more information.
Gas gangrene or myonecrosis is a well known fatal disease caused by a number of bacteria such as Clostridium perfringens, Clostridium septicum, group A Streptococcus, Staphylococcus aureus and Vibrio vulnificus (1-4). Infections from these bacteria initiate mainly from traumatic injury, except for C. septicum where no trauma is necessary at the site of infection (5). This disease is characterized by extensive tissue damage, edema, thrombosis and fluid-filled bullae, if left untreated; the complications progress very rapidly and can lead to death (5). C. septicum is a gram-positive, spore forming, obligate anaerobic bacterium that is a member of our normal gut flora as well as of other animals (5). Historically, C. septicum infection played a leading role as the causative agent of traumatic gas gangrene on the battlefield (1). After the advent of antibiotics in the mid 1950, death from C. septicum infection was drastically reduced. Although it was once thought rare, in the recent past, C. septicum infections have increasingly been identified with non-traumatic gas gangrene in patients having pre-existing medical conditions such as colonic carcinoma, defects of the bowel, leukemia, peripheral vascular diseases, diabetes, recent surgery, skin infection/burns and septic abortions (5).
Farm animals and birds (commercial turkeys) are also very vulnerable to C. septicum infection if they are not vaccinated or not treated immediately after the onset of infection. Infection often occurs from deep puncture wounds, castration and calving injuries including navel infections in newborn calves (6). A current study reports evidence for C. septicum as a primary cause of cellulitis in commercial turkeys and is associated with substantial economic loss to turkey producers (7). Turkey cellulitis is an acute diffuse infection of the dermis and subcutaneous tissue with edema.
Pathogenesis starts from the sites with poor vascular supply, although because of pH, electrolyte and osmotic differences, the colon may promote the growth of C. septicum better than most other anatomical regions (8). One of the more aggressive progenitors of gas gangrene is that the infection progresses very rapidly with a mortality rate of approximately 79% in adults, typically occurring within 48 hours of infection. Gas gangrene proceeds via disruption of blood flow to the infected site, resulting in diminished levels of oxygen and nutrients ultimately causing premature cell death and tissue necrosis (9). Tissue necrosis then causes edema and ischemia resulting in metabolic acidosis, fever, and renal failure (9). The carbon dioxide and hydrogen produced during cellular respiration move through tissue planes, causing their separation, producing features characteristic of palpable emphysema (9).
Four toxins have been isolated from C. septicum: the lethal alpha toxin, DNase beta-toxin, hyaluronidase gamma toxin, and the thiol-activated/septicolysin delta toxin (10). Alpha toxin binds to the target cell membrane and forms a channel/pore. It is considered the major virulence factor for intravascular hemolysis and tissue necrosis and is also appeared to be the immune dominant extracellural antigen (11). Purified C. septicum alpha toxin is a valuable reagent to understand the pathophysiology of this disease, development of immune detection assays and vaccines.
The cell surface receptors for alpha toxin have been identified in the recent past (12). Using retroviral mutagenesis, a mutant CHO cell line was generated that is resistant to alpha toxin and it was found that mutations occurred on glycosylphosphatidylinositol (GPI)-anchored membrane proteins. Eventually, it was confirmed that GPI anchored cell surface proteins are the receptors for alpha toxin (12).
GPI anchored proteins have received closer attention from the scientific community recently for another reason. GPI anchoring takes place through a lipid and glycan modification of certain proteins in the endoplasmic reticulum by a multiple subunit enzyme complex known as GPI transamidase (GPIT) (13, 14). Scientists have found that several subunits of GPIT are elevated in various cancers that in turn also increase levels of certain GPI-anchored proteins on the cell surface (13, 14). GPI-anchored proteins are predicted to comprise 1–2% of translated proteins in mammals (15). Several GPI-anchored proteins identified to date are tumor antigens such as carcinoembryonic antigen, mesothelin, prostate-specific stem cell antigen, and urokinase plasminogenactivator receptor, suggesting possible roles for this class of proteins in promoting tumorigenesis (13). Scientists have used C. septicum alpha toxin to capture and identify GPI-anchored proteins from human breast cancer tissues, cells and serum for proteomic analysis (13, 14). Their data indicated that patients with cancers associated with elevated GPI transamidase, showed increased alpha toxin binding of plasma proteins indicating increased levels of GPI anchored proteins. Furthermore, their results revealed very low levels of alpha toxin binding proteins in plasma from patients with no malignant disease indicating few GPI anchored proteins are present. GPI anchored proteins present in plasma from cancers patients are potential bio-markers for cancer detection (13, 14). Investigations also revealed that alpha toxin binds with the GPI glycan region as shown by retained binding of the toxin after removal of the lipid portion of the GPI anchor. Diversity of GPI anchored proteins that bind the toxin indicates that the binding occurs via the GPI glycan without peptide requirements (13, 14). Therefore, C. septicum alpha toxin has a potential to be used as a capture device for specific GPI anchor proteins to screen and identify cancer bio-markers.
By: Suzanne Canada, Ph.D.
Tanager Medical Writing
Vaccines have been used to help control diseases for more than 200 years and are the common practice for children and adults. Childhood vaccination has substantially reduced the morbidity and mortality from infectious diseases in much of the developed world, and influenza vaccinations have reduced the impact of seasonal influenza infections.1 However, medical researchers are constantly looking for ways to improve the vaccines that are already used, and develop new ones.
Opportunities for improvement of vaccines abound. For example, although much attention is given to child vaccinations, a reservoir of infection could be eliminated through promotion of adult booster shots such as pertussis booster shots for expectant mothers and close family members, to help protect susceptible newborns. In addition, some diseases that have vaccines currently available still flourish in areas of the world where infrastructures for vaccination are poor and are too costly or cannot be delivered in their current forms.1 Researchers are still trying to develop vaccines for other important diseases, such as HIV/AIDS, malaria and leishmaniasis. Vaccines are also being developed for bacterial pathogens, such as Vibrio cholerae O1 and enterotoxigenic Escherichia coli (ETEC) that are responsible for a high proportion of diarrheal disease and death in adults and children in many countries in Africa and Asia.2
By modifying the factors included in the vaccine, researchers balance the effectiveness of the immune response with the side effects. Previously, whole cell vaccines containing whole organisms that had been chemically inactivated were the norm, but the side effects of fever and discomfort following injections were much more common. Many of the vaccines used today, including those for measles and some influenza vaccines, use live, attenuated viruses. Others use killed forms of viruses, pieces of bacteria (lipopolysaccharides), or inactivated forms of bacterial toxins, known as “toxoids.” Killed viruses, lipopolysaccharides and toxoids can evoke an immune response that protects against future infection.3 Acellular vaccines were introduced in the late 1990’s that contain either three or five key bacterial proteins and have been quite effective in protecting infants and children under four with a much lower rate of side effects.
List Labs offers several virulence factors which are used in vaccine testing. For testing C. difficile vaccines; available reagents are C. difficile Toxin A (Product #152), C. difficile Toxin B (Product #155), C. difficile Toxoid A (Product #153), C. difficile Toxoid B (Product #154), and both subunits of the Binary Toxin (Products #157 and #158). Numerous Bordetella pertussis virulence factors are available for use in testing including: Products #179, #180 or #181 Pertussis Toxin, Product #170 FHA, Product #186 Fimbriae, Product #187 Pertactin, Product #188 and #189 Adenylate Cyclase and Product #400 Highly Purified B. pertussis LPS. Anthrax vaccine testing maybe carried out using Protective Antigen (Product #171) with Lethal Factor (Product #172) in a toxin neutralization assay. Although these factors are not suitable for testing on humans, they are excellent research tools.
Inactive toxins are quite useful in making antibodies or in capturing antibodies from a vaccinated population on ELISA plates. Three of our inactivated toxins, which carry mutations in the toxin active site, are B. pertussis Adenylate Cyclase Toxoid, Pertussis Toxin Mutant, Product #184 and CRM197, a non-toxic Mutant Diphtheria Toxin, Product #149. Toxoids made by formaldehyde treatment of toxins include versions of C difficile Toxins (Products #153 and #154), Diphtheria Toxoid (Product #151), Staphylococcus aureus Enterotoxoid B (Product #123), Tetanus Toxoid (Product #191) and Toxoids of Botulinum Neurotoxins A and B (Product #133 and #139, respectively).
What Are Toxin Neutralization Assays & How Do They Work?
For clinical detection or vaccine testing, it is hard to beat a toxin neutralization assay. Toxin neutralization assays (TNA) assess the ability of antibodies to protect cells in culture from the cytotoxic affect of the specific toxins. Interestingly, these assays may be used for sensitive and reliable testing for disease states where toxins are involved, as well as for development of vaccines to treat infectious disease. In TNA testing, potential sources of toxin and antibodies are combined and applied to cell culture in a series of dilutions. Excess toxin in the sample, not neutralized by the antibody, will kill the cells, the amount of excess toxin determined by the dilution of the sample which will cause a defined amount of cell death. The end point in such assays is cell viability, and this may be visualized by several different methods. A commonly used method is to visualize viable cells through metabolism of a staining reagent; the intensity of the developed color is directly proportional to the percent of remaining cell viability. TNA assays can also be used as a definitive identification of the causal agent of the disease.
TNA Assays for Clostridium Difficile Diagnosis
Cytotoxin neutralization (CTN or TNA) assays have great value in the specific diagnosis of C. difficile. Laboratory diagnosis is described by Alfa and Sepehri (Alfa, 2013). These assays can progress through a stepwise process starting with testing for glutamate dehydrogenase (GDH) in stool from potential C. difficile infected (CDI) patients. C. difficile GDH (cdGDH) is a highly active enzyme which can be readily detected and correlates well with C. difficile infections. Test results that are negative for GDH can identify samples in which C. difficile is highly unlikely, whereas tests positive for this enzyme can be used to identify potential C. difficile infections. However, since GDH is also produced by other inhabitants of the digestive tract, the presence of GDH is not conclusive evidence of C. difficile. To take diagnosis a step farther, immunological assays for C. difficile toxins A and B are used and when positive, identify C. difficile infections. Low sensitivity of these assays produce false negative results when only a small amount of toxin is present; this is when a TNA assay on highly sensitive cells comes into play. Depending on the type of cell culture, it is possible to detect C. difficile toxin B at a concentration of picograms per ml. Because cell cultures may be killed by a variety of components in a test sample, specific identity of the toxin relies on the use of standard neutralizing antibodies directed uniquely to C. difficile toxin A or B. When the antibody protects the cells from toxin directed death, the presence of, for example, the C. difficile toxin B is shown; this is a positive indication of a CDI patient. Toxin neutralization is a valuable assay in identifying patients infected with C. difficile and List Labs products are used in the development of these assays and subsequent testing to detect the toxins in samples. Products available from List Lab are C. difficile toxin A, C. difficile toxin B, our new product C. difficile GDH as well as antibodies directed to these three proteins, all of which are used to perform TNA.
TNA Assays Used to Evaluate Potential Vaccines
Another equally important use of toxin neutralization is in testing for the evaluation of potential vaccines. A paper published in 2013 by Xie et al describes a TNA developed for the evaluation of hyperimmune sera raised in animals against potential C. difficile toxin A (TcdA) and toxin B (TcdB) toxoid vaccine candidates. The authors optimized the assay using Vero cells for detection of neutralizing antibodies and for the determination of toxin potency.
TNA Assays for Anthrax Vaccines
Similar toxin neutralization assays have been developed and optimized for anthrax vaccines. These assays have been in use for over 10 years. A good review of these assays using different types of cultured cells to measure antibody levels created in response to different vaccines was provided by Ngundi et al, 2010. Anthrax toxin products available from List Labs are protective antigen (PA), lethal factor (LF), edema factor (EF), as well as the respective antibodies, all of which are used in these assays.
Alfa et al (2013) Combination of culture, antigen and toxin detection, and cytotoxin neutralization assay for optimal Clostridium difficile diagnostic testing. Can J Infect Dis Med Microbiol 24(2) 89-92. PMCID 3720004
Ngundi et al (2010) Comparison of Three Anthrax Toxin Neutralization Assays. Clinical and Vaccine Immunology 17(6) 895–903. PMID: 20375243
Xie et al (2013) Development and Optimization of a Novel Assay to Measure Neutralizing Antibodies Against Clostridium difficile Toxins. Clinical and Vaccine Immunology 20(4) 517-525. PMID: 23389929