Dr. Jekyll and Mr. Hyde: a Short History of Anthrax

Molecular Aspects of Medicine 30 (2009) 347–355

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Molecular Aspects of Medicine

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Review Dr. Jekyll and Mr. Hyde: A short history of anthrax

Maxime Schwartz *

Institut Pasteur, 25 Rue du Docteur Roux, 75015 Paris, France article info abstract

Article history: The anthrax letters crisis, following the discovery of a major bacterial warfare program in Received 29 June 2009 the USSR and the realization that Irak had been on the verge of using anthrax as a weapon Accepted 29 June 2009 during the first Gulf war, had the consequence of putting anthrax back on the agenda of scientists. Fortunately, although it was mostly unknown by the public before these events, it was far from unknown by microbiologists. Already mentioned in the bible as a disease of Keywords: herbivores, it remained a major cause of death for animals all over the planet until the end Anthrax of the 19th century, with occasional, sometimes extensive, contamination of human Bacillus anthracis beings. The aetiological agent, Bacillus anthracis, was identified by French and German Bioterrorism Bacterial warfare scientists in the 1860s and 1870s. This was the first time that a disease could be attributed Bacterial spores to a specific microorganism. The discovery by Koch that this bacterium formed spores Pathophysiology greatly contributed to the understanding of the disease epidemiology. Studies on the Bacterial toxins pathophysiology of anthrax led to the identification of two major virulence factors, the Vaccines capsule, protecting the bacilli against phagocytosis, and a tripartite toxin. The latter consists of two toxins with a common component (protecting antigen, PA) that allows the binding to and penetration into cells of two enzymes, the oedema factor EF, a calmodulin dependent adenylate cyclase, and the lethal factor LF, a specific zinc metalloprotease. The primary targets of these toxins would seem to be cells of innate immunity that would otherwise impair multiplication of the bacilli. If detected early enough, B. anthracis infections can be stopped by using antibiotics such as ciprofloxacin. Infection of animals can be prevented by the administration of vaccines, the first of which was developed by Pasteur after an historical testing at Pouilly-le-Fort which marked the beginning of the science of vaccines. Ó 2009 Elsevier Ltd. All rights reserved.

Contents

1. The comeback...... 348 2. Anthrax, an animal disease already known by Moses is still with us today ...... 349 3. Discovery of the aetiological agent ...... 350 4. Pathophysiology of anthrax and virulence factors ...... 351 5. Cure and prevention of B. anthracis infections ...... 352 6. Anthrax today...... 353 References ...... 354

* Tel.: +33 144389338. E-mail address: [email protected]

1. The comeback

On that Saturday, Robert Stevens, 63 years old, felt unusually tired. The date was September 28, 2001. Stevens lived in Florida, and worked at Boca Raton for Sun, a supermarket tabloid owned by American Media Inc. On Sunday he felt feverish. On Monday his fever had increased, he vomited and started to be disoriented. On Tuesday, he was admitted to the hospital. Having diagnosed meningitis, the doctors performed a lumbar puncture. In the cerebrospinal fluid, to their great surprise, they found a bacterium that looked like... Bacillus anthracis. Their surprise was great because human anthrax had become exceedingly rare in the United States. Furthermore this disease had only been found among individuals who had contacts with diseased animals or with products of animal origin, for instance in the leather or wool industry. And this was not the case for Robert Stevens. On October 4, the bacterium was confirmed as B. anthracis, and Robert Stevens died the day after, six days after the first symptoms. But already on October 1st, another employee of American Media Inc. had fallen ill, with symptoms similar to those of Robert Stevens. Laboratory analyses showed that he, also, had been infected with B. anthracis. Fortunately the doctors, alerted by Stevens’ case treated him immediately with high doses of ciprofloxacin, an antibiotic especially active on that bacterium, and he survived. On October 7, looking for a common source of infection of these two employees working in the same building, investigators from the FBI found spores of B. anthracis on Stevens’ computer keyboard. An enquiry revealed that, on September 19, Stevens had received a strange letter containing some white powder. This letter must have contained the spores. From then on started the anthrax letters crisis, with anxiety and then panic increasing by steps all over the country (Cole, 2003). Starting on October 12, seven individuals from the news media contracted cutaneous anthrax in New York. The week after, on October 15, an envelope containing spores of B. anthracis was found in the mail addressed to senator Daschle. The Senate’s mail room was contaminated and 28 employees working in the building were found to carry spores in their nasal cavity. The 6000 people who worked in the building were treated with ciprofloxacin. The building was closed, and its decontamination took three months. After the news media and the politicians, the postal services were stricken next. Four employees in the postal center at Brentwood, where the mail for Capitol Hill was sorted, caught anthrax, and two of them died. At this point, panic was at its most. If sorting centers were contaminated, anyone might receive spores in its mailbox. As a matter of fact, two women, one in Manhattan and the other in Connecticut, were to be infected by spores presumably received through contaminated mail, and both were to die. From the point of view of public health, this crisis was a minor event: only five persons died. However, it provoked a deep traumatism both in the USA and elsewhere (Inglesby et al., 2002). Initially, several signs had suggested that this attack, that started a week only after the destruction of the World Trade Center, bore the mark of Al Qaeda. But evidence soon accumu- lated that the sender might be instead an American scientist having access to laboratories working on B. anthracis. Seven years after the facts, on July 29, 2008, Bruce Ivins, one of the most prominent specialists in anthrax, who had turned to be highly suspected by the FBI, committed suicide (Dance, 2008; Enserinck and Bhattacharjee, 2008; Meselson, 2009). Was he the culprit? It may be so, but skeptics remain. At any rate, whoever sent the letters, the crisis showed that anthrax spores could be used as a weapon by terrorists, and that even if it did not make many victims, it could easily disorganize a whole country. This sudden awareness induced the governments of many countries to devote major efforts to the prevention of what was soon called ‘‘bioterrorism”. A measure of the trauma experienced by the American Government was reflected in the budget of the NIAID, the institute specialized in infectious diseases. This institute benefited from a budget increase of $1.5 billion in 2003 to develop studies aimed at preventing or combating bioterrorism. This increase was 2.5 greater than that obtained by that same institute between 1985 and 1998 to try and control AIDS! If it took the anthrax letters to popularize the notion of bioterrorism, the use of microorganisms as weapons of war had a long history. One that started before the very existence of microorganisms was known (Miller and Engelberg (2001), Guille- min (2005), Clunan et al. (2008), Hudson et al. (2008), Franz (2009)). Hence, for instance, the famous episode where Tatar soldiers, in 1346, sent the corpses of plague victims over the walls or the besieged town of Caffa, in Crimea. It is said that, by fleeing that town and sailing away, inhabitants of this town were responsible for starting the terrible epidemics of black plague that devastated Western Europe during the middle of 14th century. Focusing on anthrax, research on its use as a weapon was active during World War I by combatants on all sides. Then, in 1945, the soviet troops discovered close to Harbin, in Manchuria, a part of China that had been occupied by the Japanese during the war, a military installation named ‘‘Unit 731”, specialized in a secret program for the development of bacteriological warfare. Anthrax, together with cholera and plague were among the diseases they had studied in the 1930s, exper- imenting on people used as guinea pigs, essentially Chinese but also prisoners of war of American or British nationalities. In addition, the Japanese had actually used bacteriological weapons in military battles with the Chinese (Guillemin, 2008). Fol- lowing that example, several nations of the western world, including the UK, Canada and then the United States, developed bacterial warfare programs in the post-war period. However, no program took such dimensions as that developed secretly in the former USSR and revealed in the early 1990s by defectors such as Ken Alibek (Alibek and Handelman, 1999). There, under the denomination of ‘‘Biopreparat”, the Soviets developed a gigantic network of production factories and research centers spread over 40 different sites and employing up to 30,000 persons. The aim of this program was to produce lethal microorganisms, B. anthracis more than any others, in M. Schwartz / Molecular Aspects of Medicine 30 (2009) 347–355 349 quantities several times greater than would be necessary to destroy life on the whole planet. A first clue as to the existence of this program came from an accident that occurred in 1979 in a military base close to the town of Sverdlovsk (now Iekater- inbourg) in Oural (Guillemin, 1999). According to the first reports, a thousand people were said to have died of anthrax in that area. The official version was that this epidemic resulted from the consumption of contaminated meat. However, several experts had doubts which were confirmed in 1992 when President Boris Eltsine publicly acknowledged that the epidemic in Sverdlovsk had resulted from an accident in a military installation. In fact, as revealed by Ken Alibek that same year, the accident had occurred in a unit where large quantities of anthrax spores were being prepared, for potential use in rockets. An employee had forgotten to put a filter back in place in the air evacuation system, such that the spores were disseminated in the neighboring country. The victims, animal or human, were all on the path of the dominant wind from the military installation (Meselson et al., 1994). The exact number of human victims was never known for sure but must have been of a hundred at least. Knowledge about the soviet program as first revealed by the Sverdlovsk accident, the anthrax letter crisis, and also the evidence that Saddam Hussein had developed his own program in Irak before the first Gulf war, popularized B. anthracis spores as potential weapons of mass destruction. As a result, anthrax, which claims only few human victims each year, has become one of the most popular infectious diseases, whereas its very existence had remained essentially unknown to the public at large before the 1990s. It was ignored by the public but not by microbiologists and veterinarians (for recent reviews, see Turnbull (1990), Mock and Fouet (2001), Koehler (2002), Hudson et al. (2008), where many additional references can be found). Indeed, anthrax was a disease with a very long history (Klemm and Klemm, 1959).

2. Anthrax, an animal disease already known by Moses is still with us today

Anthrax is primarily a disease affecting herbivores. As such, it has been known for a very long time. Perhaps the first re- cord of anthrax is in the Bible in Exodus, chapters 7–9. ‘‘The hand of the Lord will fall on your livestock in the fields, on horses, asses, camels, herds, and flocks, with a very severe murrain.” Such were the words of Moses, about 1491 B.C., to Pharaoh who refused to let his people leave Egypt. The warning was fulfilled and the resultant fifth plague was most likely anthrax. So was probably the ‘‘burning wind of plague” that begins Homer’s Iliad, referring to events that were supposed to have occurred about 1190 B.C. The famous Greek doctor Hippocrates (5th century B.C.) actually coined the name ‘‘anthrax” (coal in Greek language), for a disease characterized by the black colour of skin lesions and of the blood. Finally, the Roman poet Virgil (70-19 B.C.) provided one of the earliest and most detailed descriptions of an anthrax epidemic in his Georgics, noting that the disease could spread to humans (Dirckx, 1981). He also recommended that one should kill the sick animals rather than try to cure them. Anthrax remained a major cause of death for animals, all over the planet, until the end of the 19th century. In Europe, it was covered in a 10th century collection of veterinary writings, the ‘‘Hippiatrika”, and again in an 11th century work, ‘‘The medicine of quadrupeds” and major episodes were recorded in reports from most European countries (Klemm and Klemm, 1959). Anthrax epizootics were responsible for enormous domestic livestock losses in Europe from the seventeenth to the 19th century. Hence, in the middle of the 18th century, a major epidemic destroyed about half of the ovine population in Europe and took a similar heavy toll in some parts of France one century later. Chabert was the first, in 1780, to give a clear description of the disease in animals (Wistreich and Lehman, 1973; Turnbull, 1990), and Barthelemy to demonstrate the transmission of the disease by inoculating blood from infected animals into healthy animals (Wilson and Miles, 1975). A striking feature of the disease is its suddenness. Even though some animals die in a few minutes without having shown any symptom, usually symptoms appear one or two days before death. After a short period of excitation, the animals look depressed, display signs of cardiac and respiratory failure, their mucosa become hemorrhagic, their throat and abdomen are swollen and blood leaks from the different body orifices. The blood is black and fails to coagulate. Although anthrax is primarily a disease of herbivores, all mammals, including humans are susceptible. In humans, the disease can take three forms, depending on the route of entry of B. anthracis spores, cutaneous, gastrointestinal, or pulmonary. Cutaneous anthrax, first described by Maret in 1752 and Fournier in 1769, usually occurs among people who have contacts with diseased animals, primarily farmers and veterinarians, the contamination occurring through skin lesions. The disease manifests itself with black scars, which, if untreated, may result in fatal septicaemia. Pulmonary anthrax, which caused the death of the five victims of the anthrax letters, had long been known, especially as the ‘‘wool-sorter disease”. If untreated, it rapidly leads to death. As for gastro-intestinal anthrax, due to the absorption of meat from diseased animals, it caused considerable human fatalities simultaneous with anthrax epidemics in animals. Thus, for instance, in 1613, the disease made about 60,000 human victims in southern Europe. In 1958, the WHO estimated the annual incidence of human cases of anthrax worldwide to be between 20,000 and 100,000 (Raffel, 1961). Anthrax is not only a disease of the past. It is still with us today, and not only as a potential weapon for bioterrorists. In developed countries, due to the application of adequate prophylactic measures, it is only sporadic. In contrast, in devel- oping countries, anthrax may still represent a major problem, for animals as well as for human beings (Hugh-Jones (1999), Hugh-Jones and Blackburn (2009)). A massive outbreak occurred in Zimbabwe during the period 1978–1980, which caused 350 M. Schwartz / Molecular Aspects of Medicine 30 (2009) 347–355

9711 human cases with 151 deaths (WHO, quoted by Turnbull). More recent examples are the epidemics in Kyrgyzstan and Zimbabwe. In the former, large but unknown numbers of animal cases have been accompanied by at least 50 human cases in 2008. In Zimbabwe, in 2008, anthrax added its toll to the severe epidemic of cholera in a totally disorganized country where actual numbers of diseases victims are difficult to ascertain; WHO reported some 200 human cases with 8 confirmed deaths, these figures being presumably underestimated (Promed, January 13, 2009).

3. Discovery of the aetiological agent

The identification of the aetiological agent of anthrax holds a very special place in the history of medicine. It was the first time that an infectious disease could be attributed to a given microbe. The observation of bacteria in the blood of animals that had just died from the disease was made independently, around 1850, both in France, by Delafond (who may have seen them as early as 1838) and Davaine and in Germany by Pollender (Klemm and Klemm, 1959; Bazin, 2008). At that time, however, none of these scientists thought that these minuscule organisms could kill a sheep or a cow. Things changed in 1863 when Davaine, having read the works of Pasteur on fermentation and putrefaction, which showed that microorganisms could produce effects out of proportion with their size, started to wonder whether this might not be so. He then proceeded to show that the injection of a droplet of blood from a diseased animal, and containing what he called the ‘‘bactéridies charbonneuses” transmitted the disease to a previously healthy animal (Davaine, 1863). Furthermore, he showed that the infec- tivity was lost upon filtration of the infectious blood, indicating that it resided in particles, presumably the ‘‘bactéridies”. However, the time was not ripe and Davaine could not convince the scientific community that the ‘‘bactéridies” were the cause of anthrax. The demonstration that such was the case was offered by Robert Koch, in his historical 1876 paper, and reinforced by Louis Pasteur in 1877. As recalled in a recent review (Hudson et al., 2008), Robert Koch’s ‘‘elegant work was the first to demonstrate unequivocally that a rod-like microorganism was consistently present in blood and tissue of diseased animals; that spores developed under starvation conditions; that these spores could transform into the rod-like bacilli under nutrient-rich conditions; that the rod like organisms could be cultured in pure form; and finally that the cultured material, either in the form of rod-like microorganisms or spores, caused anthrax disease in experimentally infected animals” (Koch, 1876; Brock, 1988). For this bacterium, Koch used the name Bacillus anthracis, given to it by Cohn in 1875. In Koch’s experiments, the bacteria were cultivated in a very small volume of medium and he only performed a limited number of subcultures, such that one could not exclude that some hypothetical component of the blood may have been carried over from the original drop and was responsible, instead of the bacteria, for transmitting the infection to the inoculated animal. This is where Pasteur performed the definitive experiment, by doing a series of subcultures in large volumes, performing what amounted to the dilution of a drop in an ocean, where all elements present in the initial drop of blood were lost, except for the bacterium that multiplied between each dilution step (Pasteur and Joubert, 1877a; Duclaux, 1896). The work on anthrax led to the formulation of what is usually referred to as the ‘‘Koch postulates” although they were originally formulated by Klebs (Brock, 1988), the rules to follow for the identification of the agent responsible for a disease:

– Reproducible identiﬁcation of the agent in diseased tissues. – In vitro culture of the agent in pure form. – Transfer of pure culture material to elicit the disease in experimental animals.

As mentioned above, an early observation of Koch was that, as had been described for other bacteria (Pasteur, 1863, 1870; Cohn, 1876), B. anthracis yields spores under starvation conditions. This, he understood, could play a major role in the epidemiology of the disease. Indeed, there existed fields, said to be ‘‘cursed with anthrax”, in which flocks could not graze without a good number of animals contracting the disease. Spores of bacteria excreted by diseased animals or coming from their cadaver presumably remained in such fields for a very long time and later infected other animals. Pasteur actually showed the presence of the bacterium in the ‘‘cursed fields” and obtained evidence that earthworms could play a role by bringing back to the surface spores present in a buried animal cadaver (Pasteur, 1880). Edmond Nocard, a veterinarian collaborator of Pasteur, described another means whereby spores could contribute to the dissemination of the disease: He wrote of ‘‘the imprudence of a new farmer, full of zeal and initiative and wishing for higher yields, who (unheard of in that area) during his first year bought a large quantity of artificial fertilizer. His wheat was superb, but the following year, no sooner had he put his flocks to pasture on the fertilized ground than anthrax appeared, and that very year he lost nearly a quarter of his stock; subsequently the disease continued to wreak havoc”. The fertilizer had been manufactured by a large company that pro- cessed animal corpses from miles around and that paid little attention when it turned them into fertilizer. From this, Nocard drew conclusions about the probable role in the spread of anthrax of the use of artificial fertilizers that had been badly prepared from infected animal wastes (Nocard, 1881). Another means whereby spores might be disseminated is by insect vectors. Amusingly, this is a concept that was apparently widespread at the beginning of the 20th century since a well known French comedy writer, Georges Feydeau, in his play ‘‘Mais n’te promène donc pas toute nue” (Don’t go walking around naked), stages a woman terrified because, having been bitten by a wasp, she is afraid that the insect might have transmitted her anthrax! One century later, even though there is M. Schwartz / Molecular Aspects of Medicine 30 (2009) 347–355 351 evidence that the transmission of spores of B. anthracis by some insects, especially tabanids, might be responsible for the spreading of the disease over rather large areas, this remains to be demonstrated conclusively (Sen and Minett (1944), Hudson et al. (2008), Hugh-Jones and Blackburn (2009)). A role of scavenging birds in the dissemination of anthrax has also been considered, but never established clearly (Turnbull, 1990, Hugh-Jones and Blackburn (2009)). Spores (Driks (2009)) are extremely resistant to a variety of physical treatments and can survive for a very long time in natural (Manchee et al., 1981) or laboratory (Jacotot and Virat, 1954) environments. Viable spores were even found in the bones of animals that died 200 years ago in Kruger National Park (Hudson et al., 2008). Clearly, the fact that B. anthracis easily yields spores with a high resistance to external conditions makes it a microorganism of choice as a biological weapon. Knowledge of the role of spores in the dissemination of the disease led to the formulation of prophylactic rules that turned out to be extremely efficient in decreasing the incidence of the disease. The cadavers of animals that die of anthrax, or suspected of it, have to be disposed in such a way that they cannot be the source of further infections.

4. Pathophysiology of anthrax and virulence factors

For Pasteur, the pathophysiology of anthrax was very simple. The bacillus grew in the blood and, since it was aerobic, it depleted the oxygen, thus asphyxiating the red blood cells (Pasteur and Joubert, 1877b). However, Pasteur was only seeing the last step, that of septicaemia, during which, indeed, asphyxia of the red cells is probably a major cause of death. What happened before during the infection? How did the bacillus manage to overcome all the mechanisms of defence that operate in mammals? In other words, what are the virulence factors of the bacillus and how do they operate in the infected host? In 1884, already, Metchnikoff was attempting to solve this question (Metchnikoff, reprinted in English in 1984). After studying the interaction of phagocytes with the anthrax bacillus, he concluded that ‘‘a battle rages between the two, which ends in favour of the phagocytes when they are able to devour the greater part of the bacteria, while the bacteria emerge victorious if the phagocytes cannot attack them.” For B. anthracis, two main virulence factors have been uncovered: the capsule and a tripartite toxin (Reviewed in Mock and Fouet, 2001). The presence of a capsule in B. anthracis was first noted by M’Fadyean (1904). Preisz (1909) then showed that animals dying of natural anthrax had numerous capsulated bacilli in their blood and tissues, whereas cultures made from those tissues showed only bacilli devoid of capsules, and he took this as evidence that the capsule was an important factor allowing the bacterium to multiply within the host. This role of the capsule as a virulence factor later received abundant confirmation. First, culture conditions were found that allowed the bacterium to produce its capsule as it does in vivo: they involved the presence of serum in the culture medium and incubation under at 5–20% CO2. Colonies of capsulated bacteria presented a characteristic ‘‘smooth” appearance. Then, ‘‘rough” variants isolated under these conditions had lost both their capsule and their virulence. Unlike the capsule identified in other pathogenic bacteria such as Pneumococci and made of polysaccharides, the capsule of B. anthracis is a linear polymer of c-D-glutamic acid. In his review written in 1961, Raffel states: ‘‘Thus the ability of the anthrax bacillus to establish itself in the tissues of the host seems a priori dependent upon the possession of the capsule, and this appears to hinge upon three known properties of this polypeptide structure. First, it can discourage the phagocytic activity of leucocytes as judged by in vitro tests; second, it can neutralize the activity of an anthracidal factor normally present in tissues; and third, it can enhance the ability of small numbers of anthrax spores to initiate infection.” In a recent review (Hudson et al., 2008), one can read: ‘‘The capsule, a linear polymer of c-D-glutamic acid, is considered the (...) major virulence factor of B. anthracis. The capsule contributes to pathogenicity by enabling the bacteria to resist phagocytosis by macrophages.” Therefore, there is no doubt that the capsule is essential for virulence. But as to how exactly the capsule protects the bacterium from phagocytosis, very little can be found in the literature (Keppie et al., 1963). In contrast, the other virulence factor, the tripartite toxin, has been and still is the object of major attention (Moayeri and Leppla, 2009). Interestingly, the idea that the bacterium could secrete a molecule involved in pathogenesis was already mentioned by Pasteur in 1877 (Pasteur and Joubert, 1877b). Indeed, he noted that filtrates of the blood from diseased animals induced the agglutination of red cells in the blood from healthy animals. Therefore, he suggested that this agglutination, a well known symptom in animals dying from anthrax, was caused by a ‘‘diastase” (old name for enzyme) produced by the bacteria. Whether this agglutinating compound had anything to do with the B. anthracis toxins as known today is not clear, but the idea was certainly fertile and found materialization in the discovery of diphtheria toxin by Pasteur’s collabo- rators, Roux and Yersin (Roux and Yersin, 1888). Furthermore, Louis Marmier, a student of Emile Roux, reported in 1895 the presence of a toxic substance in cultures of the anthrax bacillus (Marmier, 1895). A few years later, Bail and Weil found a soluble toxic substance in extracts of anthrax lesions (Bail, 1904; Bail and Weil, 1911). In the late 1940s, Cromartie et al. confirmed the production of this toxin in vivo (Cromartie et al., 1947). The final steps were made by Smith and his associates in a series of outstanding studies where they succeeded in isolating and characterizing the nature and activities of the proteinaceous complex appearing in the plasma and exudates of infected animals (Harris-Smith et al., 1958; Smith, 1958; Stanley and Smith, 1963). They showed the complex to be composed of three protein factors: protecting antigen (PA, 83 kDa), oedema factor (EF, 89 kDa) and lethal factor (LF, 90 kDa). Independently, these three factors are innocuous. However, intravenous injection of PA + LF provokes death of experimental animals, whereas intradermal injection of PA + EF produces oedema in the skin. 352 M. Schwartz / Molecular Aspects of Medicine 30 (2009) 347–355

Not much more happened in the field until the beginning of the 1980s (Stephen, 1981), when interest in the anthrax toxins was renewed, mainly because of two major discoveries. The first was the work of Gill on cholera toxin which provided a new conceptual framework to think about complex toxins (Gill, 1976; Gill et al., 1981). From that work evolved the notion that one subunit of a toxin (the so-called B subunit) could be involved in the transport of another subunit, the active component (the A subunit), into the target cell. For the anthrax toxins, subunit PA thus appeared as a likely candidate for a component involved in the specific binding to the target cell and the transport into it of both LF and EF (Singh et al., 1991). The second discovery was that of Leppla, who showed in 1982 that EF, like the toxin of Bordetella pertussis (Wolff et al., 1980), was a calmodulin-dependent adenylate cyclase causing a large increase of cytosolic c-AMP (Leppla, 1982, 1984). This was to be the first bacterial toxin whose intracellular activity could be video-imaged (Dal Molin et al., 2006). It then took more than 15 years after Leppla’s findings to establish that LF is a zinc metalloprotease which cleaves specifically the amino N terminus of mitogen-activated protein kinases (MAPKKs) (Duesbery et al., 1998; Vitale et al., 1998, 2000). The precise role of the two toxins in anthrax pathology remains to be fully elucidated. Earlier studies suggested that the toxins were responsible for death (Keppie et al., 1955; Smith et al., 1955), but recent acquisitions indicate that their primary targets are cells of innate immunity that would otherwise impair the multiplication of the bacilli (Hudson et al. (2008), Tournier et al. (2009)). They do so by altering the c-AMP and MAPK signalling pathways which are essential for activation of immune cells. The two toxins would then act on many different cell types when they reach high concentrations in the final stages of the disease following the widespread dissemination of B. anthracis in the infected animal. Before closing this section on pathophysiology, it may be worth recalling an early observation made by Pasteur and deal- ing with the now popular concept of ‘‘species barrier”. Having observed that birds were refractory to anthrax, Pasteur hypothesized that this could result from the somewhat higher body temperature of these animals, 38–42 °C, as compared to mammals. Indeed, he showed that, by placing hens for some time in cold water he rendered them sensitive to anthrax (Pasteur et al., 1878). This experiment provided a first illustration of the concept of species barrier and of its relative character (Beyer and Turnbull (2009)).

5. Cure and prevention of B. anthracis infections

Today, we know that antibiotics such as ciprofloxacin are very efficient in controlling B. anthracis infections. This is not the place to discuss antibiotics. However, it may be worth recalling that, historically, as mentioned by Howard Florey, one of the inventors of penicillin (Florey et al., 1949), anthrax provided the first scientifically reported example of ‘‘antibiosis”, a concept that led to the discovery of antibiotics. Indeed, Pasteur and Joubert showed that the injection of other – unidentified - bacteria together with B. anthracis into laboratory animals prevented them from dying with anthrax (Pasteur and Joubert, 1863). From this observation, they concluded: ‘‘These facts may entitle us to the greatest hopes for therapeutics”. The mechanism of this first reported example of bacterial antagonism is not clear but it inspired several other workers, including Ern- est Duchesne, who showed in 1897 that the injection of a culture supernatant of Penicillum glaucum rescued guinea pigs infected with pathogenic bacteria (Duchesne, 1897). If Duchesne had not died prematurely, he might have discovered penicillin 30 years before Fleming! Aside from antibiotics, additional methods are being investigated to control B. anthracis infections, for instance to prevent the action of toxins, in case bioterrorists would use an antibiotic-resistant strain, or to try and rescue an individual in whom bacteraemia would have already reached a high and potentially lethal level (Hudson et al., 2008). If the contribution of Pasteur to the discovery of antibiotics remained anecdotic, his invention of a vaccine against anthrax, based on the use of an attenuated form of the bacillus, marked the history of medicine. His extraordinary success at Pouilly-le-Fort in 1881 (Pasteur et al., 1881b), followed shortly, in 1885, by the first vaccination of a human being against rabies, marked the beginning of the science of vaccines. Admittedly, the priority of Pasteur in inventing the anthrax vaccine has been disputed (Geison, 1995). On the one hand, it is now clear that another French scientist, Henry Toussaint, well known to Pasteur, and exploiting his results on chicken cholera, succeeded in immunizing sheep against anthrax a few months before Pasteur did (Toussaint, 1880; Bazin, 2008). On the other hand, a British veterinarian, William Smith Green- field, succeeded one year before Pasteur in attenuating B. anthracis and using the attenuated bacterium to protect animals against the virulent microbe (Tigertt 1980); however, unfortunately for him, his experiments, performed at a small scale, went essentially unnoticed, even though they were published in a series of letters to Lancet in 1880 and 1881. Whatever happened at that time, and whether Pasteur’s experiments were or not performed independently of those performed by Toussaint or Greenfield, his experiments were the ones that became known worldwide and exploited for the vaccination against anthrax. In a country as distant from France as Australia, it is estimated that the introduction of vaccination in 1890 led to the protection of about 8 million sheep against anthrax between 1894 and 1900 (Todd, 1992). In the USA, the first distribution of vaccine occurred in 1897–1898. According to the ‘‘Bureau of Animal industry”, 127,369 animals were vaccinated. Whereas the average loss in livestock was 14% before the introduction of vaccination, it fell to 0.54% after vaccination (Tuffier, 1930). But what was exactly Pasteur’s vaccine? It consisted of bacteria grown for several days between 42 and 43 °C in the presence of oxygen, conditions were they did not form spores and became attenuated (Pasteur et al., 1881a). However, this vaccine was far from perfect. Sometimes, it was insufficiently attenuated and killed the animals it was supposed to protect. In M. Schwartz / Molecular Aspects of Medicine 30 (2009) 347–355 353 other instances, it was too attenuated, and failed to induce protection. One should remember that the production of each batch of vaccine required the attenuation of virulent bacilli. A major improvement in the vaccine was introduced by Max Sterne in 1937–1942, who isolated a stable avirulent variant of the bacillus (Sterne, 1939). Live spores of this strain, which gave reproducible results, thereafter became the base of the anthrax vaccine used worldwide for veterinary use (Hambleton and Turnbull, 1990, 1991). In essence, the use of Sterne’s vaccine, coupled with adequate disposal of the cadavers of animals stricken by the disease, had apparently solved the economic problem posed by anthrax, and only few scientists remained interested in studying it. However, many questions were still unsolved and among them, the nature of the attenuation mechanism exploited by Pasteur and of the stable attenuated strain isolated by Sterne. This question was not solved until the early 1980s, i.e. one century after the famous experiment at Pouilly-le-Fort! In 1983, Mikesell et al. found that virulent strains of B. anthracis harboured a large plasmid (110 Mdal), which could be cured by sequential passage at 42.5 °C, the temperature used by Pasteur in his attenuation process. Cured strains were capsulated but had lost the ability to produce the toxins and, concomitantly, had lost their virulence (Mikesell et al., 1983). Two years later, Uchida et al. (1985) and Green et al. (1985) reported the presence of a second plasmid in virulent strains, whose elimination resulted in the loss of the capsule. These two plasmids, later named pXO1 and pXO2, have since been extensively studied and sequenced (Keim et al., 2009). Sterne’s strain harboured only pXO1. As for Pasteur’s vaccine, it presumably consisted of a mixture of fully virulent bacilli, containing both plasmids, and of bacilli having lost either one of the plasmids, or both. Since there was no way to control the respective proportions of these different types, the irreproducibility of the vaccine is easily understandable. But what was it exactly in these vaccines that induced immunity against anthrax? From the beginning, the study of immunity was closely linked to that of bacterial toxins, in particular diphtheria and tet- anus toxins, which were shown to induce the appearance of ‘‘antitoxin” in the serum of toxin-injected animals (von Behring and Kitasato, 1890; Roux et al., 1894). Therefore, it was believed that a similar approach could also work for anthrax. Indeed, as recalled above, a toxic substance was reported to be present in cultures of B. anthracis (Marmier, 1895), and bacteria-free oedema fluid from anthrax lesions could actively immunize animals (Bail, 1904; Bail and Weil, 1911). For some time it seemed that the immunizing substance, as was the case for Pneumococcus, might be constituted of capsular antigen. How- ever, in the late 1940s Cromartie et al., after having confirmed that filtrates of infected tissue fluids could immunize against anthrax, showed that the immunizing substance was a globulin. Gladstone (1946) reported the existence of a ‘‘protective antigen” in cell-free cultures of the bacterium. Finally, in the 1950s, the work of Smith et al. showed that this ‘‘protective antigen” was no other than the common component of the two toxins (Stanley and Smith, 1963). These notions indicate that the immunizing properties of Sterne’s strain can be largely explained as it follows: this strain carries pXO1, it produces toxin, the PA component in particular, and this induces immunity (Harris-Smith et al., 1958). How- ever, as it is acapsulated, it is eliminated by the immune system. The Sterne’s vaccine is efficient and economic and, therefore, it is widely used to protect livestock in regions where anthrax is endemic. However, some residual virulence can be observed in goats and some laboratory animals. Hence, its use as a vaccine for human use is not straightforward, even though this has been done in the some countries such as the former USSR and China (Hambleton and Turnbull, 1990; Shlyakhov and Rubinstein, 1994; Hudson et al., 2008). Why vaccines for human use? For populations at risk, in regions of high incidence of animal anthrax, for veterinarians, for workers in industries using animal skins of doubtful origin, and...for soldiers who would be potentially exposed to bacteriological attacks or populations threatened by bioterrorists. In view of the possible dangers resulting from vaccination with attenuated strains of B. anthracis, and of the immunizing properties of PA, this latter molecule has been used to elaborate anthrax vaccines for human use, both in the UK and the USA (Hambleton and Turnbull (1990), Turnbull (1991, 2000), Friedlander et al. (1999), Cybulski et al. (2009)). Such a vaccine, for instance, was inoculated to American soldiers involved the first Gulf war. Even though one controlled experiment, conducted among workers in a goat hair-spinning mill, gave satisfactory results (Hudson et al., 2008), some recent work indicates that the immunity conferred by PA alone might not be sufficient. Therefore, new vaccines, involving additional components, are being evaluated (Hudson et al. (2008), Cybulski et al. (2009)).

6. Anthrax today

The history of anthrax, as we have seen, is clearly double-faced, reminiscent of The strange case of Dr. Jekyll and Mr. Hyde, written in 1886 by Robert Louis Stevenson. Like Mr. Hyde, anthrax has brought evil on people. Not only did it kill thousands of animals and human beings since Antiquity, and still does, but it was also turned into a potentially murderous weapon for bacteriological warfare and bioterrorism. But like Dr. Jekyll, it has done a lot of good to humanity, since its study paved the way for the fight against infectious diseases. Indeed, anthrax was the first disease that could be attributed to a specific microorganism, and its study allowed Koch to devise novel staining and cultivation methods, useful for many other bacterial pathogens. In addition, the study of anthrax led to the elaboration of the Koch’s principles which are at the foundations of medical microbiology. The success of the vaccine against anthrax started the science of vaccines in general; the work of Pasteur and his colleagues on anthrax included formulation of concepts as important as antibiosis and species barrier. Moreover, the present day studies on the pathophysiology of the disease, including an investigation of the role of its toxins, have made of B. anthracis one of the best models in infectiology (Goossens (2009)). The pathogenic character of B. anthracis was first suggested by Davaine in 1863, and this bacterium is still the object of numerous studies today. No pathogenic microorganism has been studied for such a long time: close to 150 years! However, 354 M. Schwartz / Molecular Aspects of Medicine 30 (2009) 347–355 its study did not follow a continuous course. Schematically, it proceeded in four successive bursts. The first, in the late 1870s and the 1880s, was motivated by the extensive damage caused to livestock by anthrax, and the work of Koch, Pasteur and their immediate followers led to a satisfactory control of the epizootic. The second, in the late 1940s and the 1950s, accompanied the development of programs in bacteriological warfare in different countries. Most notable in that period was the work of Smith and his colleagues on the identification of the toxins. The motivation for the third burst, in the 1980s, was quite different and lacked any economic or military objective. B. anthracis appeared as it is: a very interesting model to study bacterial pathogenesis at molecular, cellular and tissue levels. Anthrax toxins provided a good example for a mechanism of trans-membrane translocation of proteins, and the discovery of plasmids pXO1 and pXO2 allowed one to study virulence factors at a genetic level. The fourth and last burst followed the anthrax letters crisis. An unprecedented support was then allocated by governments, particularly in the USA, to develop research on microorganisms that could be used in bioterrorist attacks, including B. anthracis. The result has been a proliferation of studies that have turned B. anthracis into one of the best known pathogenic bacteria (Hudson et al. (2008), this issue). The average number of publications on the subject per year has been about 10 times higher during the past few years as compared to what it was in the 1990s. In essence, the present issue of ‘‘Molecular Aspects of Medicine” consists in a review of the work that resulted from this fourth and major burst of research on anthrax.

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