Chapter 1 - The Pretherapeutic History of Botulinum Neurotoxin

Summary

Botulimum neurotoxin food poisoning (botulism) has probably afflicted humankind as long as humans have preseved and stored food. In tenth-century Byzantium, blood sausage manufacture may have been banned for this reason. Botulinum preparations were suggested to Indian maharajas as a means of assassinating enemies. Botulism outbreaks in Germany in the eighteenth and nineteenth centuries led to warnings against harmful consumption of blood sausages.

In 1820, Justinus Kerner published case histories detailing the signs and symptoms of the disease we now call botulism, and postulated a biological causative agent that developed under anaerobic condition and affected the motor and autonomic nervous systems. The bacillus Clostridium botulinum was identified by van Ermengem in 1895. multiple serological subtypes were isolated in the early twentieth century, followed by identification of wound botulism in 1950, and infant botulism in 1976. Use as a bioweapon was considered in World War I. Botulinum neurotoxin type A was isolated in the 1920s. The US government investigated its deployment in World War II. After the war, clinically therapeutic formulations were prepared in the USA and Britain.

Unintended intoxication with botulinum neurotoxin (botulism) occurs only rarely, but its high fatality rate makes it a great concern for the general public and the medical community. In the USA, an average of 110 cases of botulism are reported each year. Of these, approximately 25% are food borne, 72% are infant botulism and the rest are wound botulism. Outbreaks of food-borne botulism involving two or more persons occur most years and are usually caused by eating contaminated home-canned foods.

Botulism in Ancient Times

Botulinum neurotoxin poisoning probably has afflicted humankind through the mists of time. As long as humans have preserved and stored food, some of the chosen conditions would be optimal for the presence and growth of the toxin-producing pathogen Clostridium botulinum: for example, the storage of ham in barrels of brine, poorly dried and stored herring, trout packed to ferment in willow baskets, sturgeon roe not yet salted and piled in heaps on old horsehides, lightly smoked fish or ham in poorly heated smoking chambers and insufficiently boiled blood sausages.

However, in ancient times there was no general knowledge about the causal relationship between the consumption of spoiled food and a subsequent fatal paralytic disease, nowadays recognized as botulism. Only some historical sources reflect a potential understanding of the life-threatening effects of consuming food intoxicated with botulinum neurotoxin. Louis Smith, for example, reported in his textbook on botulism a dietary edict announced in the tenth century by Emperor Leo VI of Byzantium (886–911), in which manufacturing of blood sausages was forbidden (Smith, 1977). This edict may have its origin in the recognition of some circumstances connected with cases of food poisoning. Also, some ancient formulae suggested by shamans to Indian maharajas for the killing of personal enemies give hint of an intended lethal application of botulinum neurotoxin: a tasteless powder extracted from blood sausages dried under anaerobic conditions should be added to the enemies’ food at an invited banquet. Because the consumer’s death occurred after he or she had left the murderer’s place, with a latency of some days, the host was probably not suspected (Erbguth, 2008).

Botulism Outbreaks in Germany in the Eighteenth and Nineteenth Centuries

Accurate descriptions of botulism emerge in the German literature from two centuries ago when the consumption of improperly preserved or stored meat and blood sausages gave rise to many deaths throughout the kingdom of Württemberg in southwestern Germany. This area near the city of Stuttgart developed as the regional focus of botulinum toxin investigations in the eighteenth and nineteenth centuries. In 1793, 13 people were involved in the first well-recorded outbreak of botulism in the small southwest German village of Wildbad; six died. Based on the observed mydriasis in all affected victims, the first official medical speculation was that the outbreak was caused by an atropine (Atropa belladonna) intoxication. However, in the controversial scientific discussion, the term “sausage poison” was introduced by the exponents of the opinion that the fatal disease in Wildbad was caused by the consumption of “Blunzen,” a popular local food from cooked pork stomach filled with blood and spices.

The number of cases of suspected sausage poisoning in southwestern Germany increased rapidly at the end of the eighteenth century. Poverty followed the devastating Napoleonic Wars (1795–1813) and led to the neglect of sanitary measures in rural food production (Grüsser, 1986). In July 1802, the Royal Government of Württemberg in Stuttgart issued a public warning about the “harmful consumption of smoked blood-sausages.” In August 1811, the medical section of the Department of Internal Affairs of the Kingdom of Württemberg, on Stuttgart again, addressed the problem of “sausage poisoning,” considering it to be caused by hydrocyanic acid, known at that time as “prussic acid.” However, the members of the nearby Medical Faculty of the University of Tübingen disputed that prussic acid could be the toxic agent in sausages, suspecting a biological poison. One of the important medical professors of the University of Tübingen, Johann Heinrich Ferdinand Autenrieth (1772–1835), asked the government to collect the reports of general practitioners and health officers on cases of food poisoning for systematic scientific analyses. After Autenrieth had studied these reports, he issued a list of symptoms of the so-called sausage poisoning and added a comment, in which he blamed the housewives for the poisoning because they did not dunk the sausages long enough in boiling water while trying to prevent the sausages from bursting (Grüsser, 1998). The list of symptoms was distributed by a public announcement and contained characteristic features of food-borne botulism such as gastrointestinal problems, double vision, mydriasis and muscle paralysis.

In 1815, a health officer in the village of Herrenberg, J. G. Steinbuch (1770–1818), sent the case reports of seven intoxicated patients who had eaten liver sausage and peas to Professor Autenrieth. Three of the patients had died and the autopsies had been carried out by Steinbuch himself (Steinbuch, 1817).

Justinus Kerner’s Observations and Publications on Botulinum Toxin 1817–1822

Contemporaneously with Steinbuch, the 29-year-old physician and Romantic poet Justinus Kerner (1786–1862) (Fig. 1.1), then medical officer in a small village, also reported a lethal food poisoning. Autenrieth considered the two reports from Steinbuch and Kerner as accurate and important observations and decided to publish them both in 1817 in the Tübinger Blätter für Naturwissenschaften und Arzneykunde [Tübinger Papers for Natural Sciences and Pharmacology] (Kerner, 1817; Steinbuch, 1817).

Fig. 1.1 Justinus Kerner, 1855.

Kerner again disputed that an inorganic agent such as hydrocyanic acid could be the toxic agent in the sausages, suspecting a biological poison instead. After he had observed further cases, Kerner published a first monograph in 1820 on “sausage poisoning” in which he summarized the case histories of 76 patients and gave a complete clinical description of what we now recognize as botulism. The monograph was entitled “Neue Beobachtungen über die in Württemberg so häufig vorfallenden tödlichen Vergiftungen durch den Genuss geräucherter Würste [New Observations on the Lethal Poisoning That Occurs so Frequently in Württemberg Owing to the Consumption of Smoked Sausages] (Kerner, 1820). Kerner compared the various recipes and ingredients of all sausages that had produced intoxication and found that among the ingredients of blood, liver, meat, brain, fat, salt, pepper, coriander, pimento, ginger and bread the only common ones were fat and salt. Because salt was probably known to be “innocent,” Kerner concluded that the toxic change in the sausage must take place in the fat and, therefore, called the suspected substance “sausage poison,” “fat poison” or “fatty acid.” Later, Kerner speculated about the similarity of the “fat poison” to other known poisons, such as atropine, scopolamine, nicotine and snake venom, which led him to the conclusion that the fat poison was probably a biological poison (Erbguth, 2004).

In 1822, Kerner published 155 case reports including autopsy studies of patients with botulism and developed hypotheses on the “sausage poison” in a second monograph Das Fettgift oder die Fettsäure und ihre Wirkungen auf den thierischen Organismus, ein Beytrag zur Untersuchung des in verdorbenen Würsten giftig wirkenden Stoffes [The Fat Poison or the Fatty Acid and Its Effects on the Animal Body System, a Contribution to the Examination of the Substance Responsible for the Toxicity of Bad Sausages] (Kerner, 1822) (Fig. 1.2). The monograph contained an accurate description of all muscle symptoms and clinical details of the entire range of autonomic disturbances occurring in botulism, such as mydriasis, decrease of lacrimation and secretion from the salivary glands, and gastrointestinal and bladder paralysis. Kerner also experimented on various animals (birds, cats, rabbits, frogs, flies, locusts, snails) by feeding them with extracts from bad sausages and finally carried out high-risk experiments on himself. After he had tasted some drops of a sausage extract he reported: “… some drops of the acid brought onto the tongue cause great drying out of the palate and the pharynx” (Erbguth and Naumann, 1999).

Fig. 1.2 Title of Justinus Kerner’s second monograph on sausage poisoning, 1822.

Kerner deduced from the clinical symptoms and his experimental observations that the toxin acts by interrupting the motor and autonomic nervous signal transmission (Erbguth, 1996). He concluded: “The nerve conduction is brought by the toxin into a condition in which its influence on the chemical process of life is interrupted. The capacity of nerve conduction is interrupted by the toxin in the same way as in an electrical conductor by rust” (Kerner, 1820). Finally, Kerner tried in vain to produce an artificial “sausage poison.”

In summary, Kerner’s hypotheses concerning “sausage poison” were that (1) the toxin developed in bad sausages under anaerobic conditions, (2) the toxin acts on the motor nerves and the autonomic nervous system and (3) the toxin is strong and lethal even in small doses (Erbguth and Naumann, 1999).

In Chapter 8 of the 1822 monograph, Kerner speculated about using the “toxic fatty acid” botulinum toxin for therapeutic purposes. He concluded that small doses would be beneficial in conditions with pathological hyperexcitability of the nervous system (Erbguth, 2004). Kerner wrote: “The fatty acid or zoonic acid administered in such doses, that its action could be restricted to the sphere of the sympathetic nervous system only, could be of benefit in the many diseases which originate from hyperexcitation of this system” and “by analogy it can be expected that in outbreaks of sweat, perhaps also in mucous hypersecretion, the fatty acid will be of therapeutic value.” The term “sympathetic nervous system” as used during the Romantic period, encompassed nervous functions in general. “Sympathetic overactivity” then was thought to be the cause of many internal, neurological and psychiatric diseases. Kerner favored the “Veitstanz” (St. Vitus dance – probably identical with chorea minor) with its “overexcited nervous ganglia” to be a promising indication for the therapeutic use of the toxic fatty acid. Likewise, he considered other diseases with assumed nervous overactivity to be potential candidates for the toxin treatment: hypersecretion of body fluids, sweat or mucus; ulcers from malignant diseases; skin alterations after burning; delusions; rabies; plague; consumption from lung tuberculosis; and yellow fever. However, Kerner conceded self-critically that all the possible indications mentioned were only hypothetical and wrote: “What is said here about the fatty acid as a therapeutic drug belongs to the realm of hypothesis and may be confirmed or disproved by observations in the future” (Erbguth, 1998).

Justinus Kerner also advanced the idea of a gastric tube, suggested by the Scottish physician Alexander Monro in 1811, and adapted it for the nutrition of patients with botulism; he wrote: “if dysphagia occurs, softly prepared food and fluids should be brought into the stomach by a flexible tube made from resin.” He considered all characteristics of modern nasogastric tube application: the use of a guide wire with a cork at the tip and the lubrication of the tube with oil.

Botulism Research after Kerner

After his publications on food-borne botulism, Kerner was well known to the German public and amongst his contemporaries as an expert on sausage poisoning, as well as for his melancholy poetry. Many of his poems were set to music by the great German Romantic composer Robert Schumann (1810–1856), who had to quit his piano career because of the development of a pianist’s focal finger dystonia. Kerner’s poem The Wanderer in the Sawmill was the favorite poem of the twentieth-century poet Franz Kafka (in full in Appendix 1.1). The nickname “Sausage Kerner” was commonly used, and “sausage poisoning” was known as “Kerner’s disease.” Further publications in the nineteenth century by various authors (e.g., Müller, 1869) increased the number of reported cases of “sausage poisoning,” describing the fact that the food poisoning occurred after the consumption not only of meat but also of fish. However, these reports added nothing substantial to Kerner’s early observations. The term “botulism” (from the Latin botulus, “sausage”) appeared at first in Müller’s reports and was subsequently used. Therefore, “botulism” refers to poisoning caused by sausages and not to the sausage-like shape of the causative bacillus discovered later (Torrens, 1998).

The Discovery of “Bacillus botulinus” in Belgium

The next and most important scientific step was the identification of Clostridium botulinum in 1895–1896 by the Belgian microbiologist Emile Pierre Marie van Ermengem of the University of Ghent (Fig. 1.3).

Fig. 1.3 Emile Pierre Marie van Ermengem 1851–1922.

On December 14, 1895, an extraordinary outbreak of botulism occurred amongst the 4000 inhabitants of the small Belgian village of Ellezelles. The musicians of the local brass band “Fanfare Les Amis Réunis” played at the funeral of the 87-year-old Antoine Creteur and as it was the custom gathered to eat in the inn “Le Rustic” (Devriese, 1999). Thirty-four people were together and ate pickled and smoked ham. After the meal, the musicians noticed symptoms such as mydriasis, diplopia, dysphagia and dysarthria, followed by increasing muscle paralysis. Three of them died and ten nearly died. A detailed examination of the ham and an autopsy were ordered and conducted by van Ermengem, who had been appointed Professor of Microbiology at the University of Ghent in 1888 after he had worked in the laboratory of Robert Koch in Berlin in 1883. Van Ermengem isolated the bacterium in the ham and in the corpses of the victims (Fig. 1.4), grew it, used it for animal experiments, characterized its culture requirements, described its toxin, called it “Bacillus botulinus,” and published his observations in the German microbiological journal Zeitschrift für Hygiene und Infektionskrankheiten [Journal of Hygiene and Infectious Diseases] in 1897 (an English translation was published in 1979) (van Ermengem, 1897). The pathogen was later renamed Clostridium botulinum. Van Ermengem was the first to correlate “sausage poisoning” with the newly discovered anaerobic microorganism and concluded that “it is highly probable that the poison in the ham was produced by an anaerobic growth of specific micro-organisms during the salting process.” Van Ermengem’s milestone investigation yielded all the major clinical facts about botulism and botulinum neurotoxin: (1) botulism is an intoxication, not an infection; (2) the toxin is produced in food by a bacterium; (3) the toxin is not produced if the salt concentration in the food is high; (4) after ingestion, the toxin is not inactivated by the normal digestive process; (5) the toxin is susceptible to inactivation by heat; and (6) not all species of animals are equally susceptible.

(a) Numerous spores among the muscle fibers (Ziehl ×1000).

(b) Culture (gelatine and glucose) of mature rod-shaped forms of “Bacillus botulinus” from the ham on the eighth day (×1000).

(From van Ermengem, 1897.)

Fig. 1.4 Microscopy of the histological section of the suspect ham at the Ellezelles botulism outbreak.

Botulinum Neurotoxin Research in the Early Twentieth Century

In 1904, when an outbreak of botulism in the city of Darmstadt, Germany, was caused by canned white beans, the opinion that the only botulinogenic foods were meat or fish had to be revised. The bacteria isolated from the beans by Landmann (1904) and from the Ellezelles ham were compared by Leuchs (Leuchs, 1910) at the Royal Institute of Infectious Diseases in Berlin. He found that the strains differed, and the toxins were serologically distinct. The two types of Bacillus botulinus did not receive their present letter designations of serological subtypes until Georgina Burke, who worked at Stanford University, designated them as types A and B (Burke, 1919). Over the next decades, increases in food canning and food-borne botulism went hand in hand (Cherington, 2004). The first documented outbreak of food-borne botulism in the USA was caused by commercially conserved pork and beans and dates from 1906 (Drachmann, 1971; Smith, 1977). Techniques for killing the spores during the canning process were subsequently developed. The correct pH (<4.0), the osmolarity needed to prevent clostridial growth and toxin production, and the requirements for toxin inactivation by heating were defined.

In 1922, type C was identified in the USA by Bengston and in Australia by Seddon; type D and type E were characterized some years later (type D in the USA in 1928 by Meyer and Gunnison; type E in the Ukraine 1936 by Bier) (Kriek and Odendaal, 1994; Geiges, 2002). Type F and type G toxins were identified in 1960 in Scandinavia by Moller and Scheibel and in 1970 in Argentina by Gimenex and Ciccarelli (Gunn, 1979; Geiges, 2002). In 1949, Burgen and his colleagues in London discovered that botulinum toxin blocked the release of acetylcholine at neuromuscular junctions (Burgen et al., 1949). The essential insights into the molecular actions of botulinum toxin were gained by various scientists after 1970 (Dolly et al., 1990; Schiavo et al., 1992, 1993; Dong et al., 2006; Mahrhold et al., 2006), when its use as a therapeutic agent was pioneered by Edward J. Schantz and Alan B. Scott.

Until the last century, botulism was thought to be caused exclusively by food that was contaminated with preformed toxin. This view has changed since the 1950s, as spores of C. botulinum were discovered in contaminated wounds (wound botulism) in the 1950s and in the intestines of babies in 1976 (infant botulism) (Merson and Dowell, 1973; Pickett et al., 1976; Arnon et al., 1977). The number of cases of food-borne and infant botulism has changed little in recent years, but wound botulism has increased because of the use of black-tar heroin, especially in California.

Swords to Ploughshares

Before the therapeutic potential of botulinum neurotoxin was discovered, around 1970, its potential use as a weapon was recognized during World War I (Lamb, 2001). The basis for its use as a toxin was investigations by Hermann Sommer and colleagues working at the Hooper Foundation, University of California, San Francisco in the 1920s: the researchers were the first to isolate pure botulinum neurotoxin type A as a stable acid precipitate (Snipe and Sommer, 1928; Schantz, 1994). With the outbreak of World War II, the US government began intensive research into biological weapons, including botulinum toxin, particularly in the laboratory at Camp Detrick (later renamed Fort Detrick) in Maryland. Development of concentration and crystallization techniques at Fort Detrick was pioneered by Carl Lamanna and James Duff in 1946. The methodology was subsequently used by Edward J. Schantz to produce the first batch of toxin, which was the basis for the later clinical product (Lamanna et al., 1946). The entrance of botulinum toxin into the medical therapeutic armamentarium in Europe also came from military laboratories to hospitals: in the UK, botulinum toxin research was conducted in the Porton Down laboratories of the military section of the Centre for Applied Microbiology and Research (CAMR), which later provided British clinicians with a therapeutic formulation of the toxin (Hambleton et al., 1981).