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Zentralblatt für Bakteriologie – 100 years ago: Early considerations of the El Tor vibrios
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International Journal of Medical Microbiology 296 (2006) 333–340 www.elsevier.de/ijmm
EDITORIAL
Zentralblatt fu¨r Bakteriologie – 100 years ago: Early considerations of the El Tor vibrios The most famous advocate of the water borne transmission of cholera was John Snow (1849/1855). Already famous for being the person to introduce ether for anaesthesia in England, notably to Queen Mary (Bulloch, 1938), Snow’s work as an epidemiologist still commands attention from scholars (e.g. Eyler, 2001). In the mid nineteenth century the miasmatic theory of cholera was prevalent and editors of scientific journals were reluctant to publish his findings on the water-borne origin of the disease, so he did it at his own cost (Snow, 1849/1855). The water-borne epidemiology of cholera was confirmed by Robert Koch and the members of the ‘‘German Cholera Expedition’’ (without mentioning John Snow) when they found in 1884 the vibrios in the so-called ‘‘tanks’’ in Bengal. Tanks are water-basins (small lakes or artificially basins), surrounded by huts, and served for water-storage. The inhabitants used the water not only as sources of drinking-water, but also for bathing and washing clothes. In the small epidemic observed by Koch even the soiled clothes of the first cholera victim were washed in this peculiar tank (Koch, 1884c). Koch’s verdict, that only diseased humans or carriers were the source of new cases of cholera was one of many epochal insights but over time has had to be rethought concerning cholera in light of the movement of V. cholerae between estuarine habitats and man. Koch announced in his report from Alexandria dated 17th of September 1883, that ‘‘the mission found the constant presence of a specific bacillus in the intestinal of persons dying from cholera’’ (Koch, 1883). He concluded ‘‘that there could be no doubt that the bacillus stood in some relation to the cholera process’’ (Howard-Jones, 1984), but he had not obtained a pure culture. The isolation was announced in his fifth dispatch dated 7th of January 1884 (Koch, 1884a), and this date should be accepted as the real time of the discovery of the aetiological agent of cholera. The term ‘‘comma bacillus’’ was coined in his 6th dispatch, dated 2nd of February, when he described the germ as ‘‘a little 1438-4221/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2006.04.001
bent, like a comma’’ (‘‘ein wenig gekru¨mmt, einem Komma a¨hnlich’’) (Koch, 1884b). What Koch apparently had not seen or had ignored was a booklet published in 1854 by Filippo Pacini (1812–1883), Professor of Anatomy at Florence. There Pacini described what he had seen in cholera dejections: ‘‘miriardi di vibrioni’’ (Pacini, 1854). Coming to the conclusion that these vibrios were the causative agents, Pacini’s work was honoured by the Judicial Commission of the International Committee on Bacteriological Nomenclature, 1965, when they adopted ‘‘Vibrio cholerae Pacini, 1854’’ as the official, correct name of the cholera vibrio. Questions over the validity of Koch’s findings appeared shortly after Koch’s announcement (see von Pettenkofer, 1887; Rimpau, 1935; Anonymous, 1884a, b; India Office, 1885; Sanderson, 1885) and recent work has reconsidered the circumstances surrounding the criticisms of Koch’s work by Klein and the British Commission (Ogawa, 2000). Remarkably, the scepticism over V. cholerae as the cause of cholera persisted for almost 20 years (Howard-Jones, 1975). In part this was due to the ‘‘yseveral hundreds different species of vibrios y isolated from water, manure, putrid fluids, human dejections etc y. which are more or less similar to cholera vibrios’’ in their cultural and biochemical activities (Kolle, 1903). Examples of which are listed in Table 1. Other reasons included the problems of reproducing the disease in animal models. In order to reduce confusion in the identification of cholera-like vibrios and V. cholerae, Robert Koch, Martin Kirchner and Wilhelm Kolle wrote a pamphlet on the bacteriological diagnosis of cholera which was published in 1902 as: ‘‘Writ by the Minister of religious, educational and medical affairs, regarding instruction for the bacteriological ascertainment of cases of cholera, from 6th of November 1902’’ (‘‘Erlaß des Ministers der geistlichen, Unterrichts- und Medizinalangelegenheiten, betreffend Anleitung fu¨r die bakteriologische Feststellung der Cholerafa¨lle, vom 6. November 1902’’). The following examinations must be done in cases of
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Table 1.
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Selected Vibrio species similar to V. cholerae isolated from a variety of sources prior to 1900
Designation
Reference(s)
Isolated from
Possible species (Bergey’s Manual, 1974)a
V. V. V. V. V. V. V.
Finkler and Prior, 1884 Gamaleia, 1888 Pasquale, 1891 Pasquale, 1891 Dunbar, 1893 Iva´noff, 1893 Neisser, 1893; Gu¨nther 1893
Cholera nostras (man) Gastroenteritis in poultry Water well in Ghinda near Massaua Diarrhoea (man) River Elbe Faeces of man with typhoid fever Tap-water from the River Spree
V. cholerae biotype proteus V. cholerae biotype proteus V. choleraeb Unknown V. cholerae biotype albensisc V. cholerae varietyd V. cholerae (low virulence)? Contamination ?e
finkleri metschnikovii Ghinda Massauah phosphorescens Iwanoff Berolinensis
a
In Bergey’s Manual of Systematic Bacteriology, 1st edit., 1984, vol. 1 these ‘‘species’’ were no longer included. Not mentioned in Bergey’s. c According to Bergey’s Manual (1974) the type strain ATCC 14547 is indistinguishable from V. cholerae by DNA homology. d Bergey’s 6th ed., 1948. e Pfeiffer (1896) believed that the vibrios were not present in the stools of the patient but an external contamination. b
suspected cholera: Microscopical examination of stool (smears from flakes of the dejecta); 2. Alkaline gelatine plates; 3. Alkaline agar plates; 4. Enrichment in alkaline peptone-water; 5. Examination of pure cultures by agglutination, and 6. Pfeiffer’s reaction, i.e. serum killing of the vibrios in immune animals (Kolle, 1903). The document was also translated and published in England (Note the absence of the’’cholera-red reaction’’ (nitroso-indole reaction), introduced by Poehl in 1886 (cited by Petri, 1893) and proposed independently by Bujwid (1887, 1888) as a marker of V. cholerae). This spectrum of mostly non-specific methods helps explain why the laboratory diagnosis of cholera was so confused. Given this history of uncertainty as to the significance of the cholera-like vibrios the situation was muddied further by periodic but repeated isolation of yet more varieties of vibrio-like bacteria from pilgrims returning from Mecca. At the turn of the century (1900) there were two main routes of the pilgrimage to Mecca (also known as the Haj): pilgrims from Asia Minor, mostly Turks, landed at Yenbo before travelling by foot or camel to Medina and then Mecca (Fig. 1). The second route taken by most other nationalities (Egyptians, Algerians, Syrians, and Russians) arrived at the port of Jiddah and then firstly to Mecca. The returning pilgrims were forced to go via the quarantine camps, the closest camp under ‘‘Egyptian’’ control (rather than Turkish) was El Tor. The time taken to reach El Tor from Jiddah and Yenbo was between three to eight weeks effectively preventing any cholera entering Europe via the Suez Canal. When transportation by boat was established the time taken was reduced considerably with consequent increase in the number of pilgrims still excreting vibrios. The dreadful overcrowded conditions onboard have been described by Ruffer (1899) and make for harrowing reading. The sanitary station at El Tor was located at the West Coast of the Sinai Peninsula in ‘‘a spot of unsurpassed
attractiveness’’ (Anonymous, 1907), and was established in 1877 following discussions of the first International Sanitary Conferences at Constantinople in 1866. Several bacteriology laboratories were established at the quarantine stations, not just at El Tor but also at Alexandria, Suez and Port Said in order to investigate the pilgrims. Across the Red Sea there were further quarantine stations in the Turkish occupied region (now Saudi Arabia; Anonymous, 1907). The pilgrims had to pass through the quarantine station in numbers reaching 20,000 people at a time. The camp at El Tor was surrounded by wire-fencing ensuring that after disembarkation there was only one way to the camp – through the disinfection establishment (‘‘Desinfektionsanstalt’’). The pilgrims were made to strip, female European physicians and nurses took care for the female pilgrims, and they had to douche. Meanwhile the clothes and the personal effects were disinfected by steam or chemical methods. Then the pilgrims were sent to ‘‘sections’’ again fenced off by barbed wire (Fig. 2). If infectious diseases broke out they were restricted to this area. The length of the quarantine depended upon the epidemiological situation in the Hedjaz region. If there was no cholera or plague (‘‘pe´le´rinage net’’) the duration was three days, in the case of epidemics in the area (‘‘pe´le´rinage brut’’) 18 days (Gotschlich, 1904; Kaufmann, 1892). Immediately upon arriving in Alexandria (Sir) Marc Armand Ruffer (1859–1917), President of the Sanitary, Maritime and Quarantine council of Egypt, based at the Port Vieux Laboratory, Alexandria, Egypt, orchestrated investigations into the bacteriology of cholera and dysentery. As early as 1897 Dr. Galvani isolated a vibrio from a dead pilgrim which agglutinated with cholera antiserum (Ruffer, 1907; Bryceson, 1977). Subsequent investigations of the intestinal bacteriology of the pilgrims who had died at El Tor quarantine camp revealed a further five strains isolated from pilgrims who had been suffering with dysentery and not cholera.
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Fig. 1. Map showing the location of El Tor at the Red Sea and of other towns in the Hedjaz area.
Fig. 2. The quarantine camp at El Tor as drawn by Paul Kaufmann (1892). The sketches give no indication as to the conditions of the camp when housing 20,000 pilgrims.
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Table 2.
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Years in which cholera was recorded in Egypta
Year of epidemic/ outbreak
World Pandemic
Number of deaths
1831 1834 1837 1848 1850 1857 1865
2nd 2nd 2nd 3rd 3rd 3rd 4th
1882/1883 1895/1896 1902 1947
5th 5th 6th 7th
150,000 Similar to above ? ? ? ? 460,000 (30,000 pilgrims) 48,000–58,000 16,571–37,000 34,600 20,800
a
Compiled from (Shousha, 1947; Anonymous, 1951; Pollitzer, 1959). Ranges represent varying values in the different sources.
Cholera had not been reported that year at the Haj and did not appear there until 1902 (see Table 2). In 1903 Kolle and Gotschlich examined many other cholera-like vibrios including two strains obtained from Heinrich Bitter who had isolated these (and other strains) from pilgrims with and without cholera at El Tor (Kolle and Gotschlich, 1903). Heinrich Bitter (1863–1918) studied medicine in Munich, where he graduated MD in 1886. After a short time at the Clinic of Dermatology at the University of Wu¨rzburg, he became assistant at the Institute of Hygiene at Breslau (habilitation 1891). In 1893 he was offered the post as an ‘Inspector of Sanitary Matters’’ (Inspektor des Sanita¨tswesens) at Cairo, where he built a new Institute of Hygiene. Robert Koch asked Carl Flu¨gge for a successor for Heinrich Bitter who recommended Emil Gotschlich (1870–1949). Thus, Gotschlich became head of the Sanitary Board at Alexandria in 1896 as well as Egyptian delegate at the international ‘‘Conseil Sanitaire, Maritime et Quarantinaire’’. Emil Gotschlich had trained under Carl Flu¨gge at the Institute of Hygiene, University of Breslau. After two years there he accepted the invitation to Egypt. He lost his post at the beginning of World War I, but later became director of the Institutes of Hygiene at Halle, Saarbru¨cken, Giessen and Heidelberg (Uhlenhuth, 1930). By 1905 Emil’s younger brother Felix Gotschlich was working at the laboratories of the ‘‘Conseil sanitaire maritime et quarantenaire d’Egypt’’ in Alexandria and continuing the work on the role of cholera-like vibrios in enteric disease. Felix Gotschlich found further varieties of the comma bacillus in the intestines at autopsy from patients at the sanitation camp at El Tor. The dead men had arrived at El Tor in different ships and had died
1–13 days after disembarkation. They had not suffered from cholera (which had not been reported at the festival that year) but instead had dysenteric disease (which had been reported). Felix Gotschlich isolated 38 strains of vibrio from 6 people who had died of colitis, colitis gangraenosa or dysentery at El Tor (and two further strains the following year, 1906, under similar circumstances). Six strains were agglutinated by cholera antisera (Gotschlich, 1906). In common with Koch’s Vibrio cholerae they were positive for Pfeiffer’s reaction, had typical morphology of cholera vibrios (one flagellum), liquefied gelatine, coagulated milk, were pathogenic for guinea pigs and apathogenic for pigeons. The cholera-red reaction was positive with the exception of one strain. The latter one showed a positive reaction too after addition of some potassium nitrate to the peptone-water medium. Gotschlich did not mention the behaviour on blood agar plates. In contrast the other 32 strains of vibrios not agglutinated by cholera antiserum all showed haemolysis, but presented varying cholera-red reactions and milk coagulation. All were pathogenic for guinea pigs, a few for pigeons also. Gotschlich concluded that the six vibrios (agglutinated by cholera antisera) were ‘‘real cholera vibrios’’, and that the diseased pilgrims were ‘‘cholera carriers’’. He gave no special name to these vibrios since he was sure about their identity as V. cholerae. Gotschlich (1906) also reported that his brother Emil had examined a vibrio isolated by Ruffer in 1897 in El Tor from faeces of a pilgrim without any signs of cholera. This strain was morphologically identical with V. cholerae, agglutinated by cholera antiserum, highly pathogenic for guinea pigs, showed a positive cholera-red reaction but was negative in Pfeiffer’s reaction. This is likely to be the same organism that Ruffer credited to Dr. Galvani. Reviewing the bacteriology of Gotschlich and Dr. Zirolia, Director of the Port Said laboratory, Ruffer (1907) highlighted the uncertainty over the variation in number of flagella and, more importantly, the unreliable bacteriological diagnosis obtained by using anticholera serum since the El Tor strains reacted. As the isolates were from cases of dysentery Ruffer felt that the use of anti-cholera serum was insufficient. The vibrios were being forwarded to European laboratories on a regular basis and the vibrios studied by Felix Gotschlich were sent to the State Serotherapeutic Institute in Vienna. There, Rudolf Kraus (1868–1932) carried out further examinations of the organisms and 100 years ago in this journal Kraus, published his findings in two papers, assisted by E. Prˇ ibram and A. Prantschoff (Kraus and Prˇ ibram, 1906; Kraus and Prantschoff, 1906). Rudolf Kraus was born on 31st of October 1868 in Jungbunzlau/Bohemia (now Mlada´ Boleslaw), studied medicine in Prague and graduated there in 1893. He
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then became assistant at the Institute of Pathology in Vienna, trained under Richard Paltauf (director of the Institute of Pathology as well as of the Serotherapeutic Institute). In 1903 he habilitated in the field of general and experimental pathology, and was appointed in 1910 as Assistant Professor. In 1913 he became director of the Bacteriological Institute at Buenos Aires, later of the Serological Institute at Butantan (Sa˜o Paulo). Ten years later he came back to Vienna and succeeded Paltauf as a director of the Serotherapeutic Institute. In 1928 he was invited by the Chilean government to establish a new ‘‘Istituto Bacteriologico de Chile’’ in Santiago de Chile also becoming director general of the International Society of Microbiology in 1930. Kraus is known to microbiologists by the detection of bacterial precipitation (Ko¨hler, 1997), as well as editor of several important textbooks: the 3rd edition of ‘‘Handbuch der pathogenen Mikroorganismen’’ 1927 (together with Kolle and Uhlenhuth), ‘‘Handbuch der Immunita¨tsforschung und experimentellen Therapie’’ (together with C. Levaditi, 2nd ed. 1914), and ‘‘Handbuch der mikroskopischen Technik’’ (together with P. Uhlenhuth 1922–24). Rudolf Kraus died on 15th/16th July 1932 in Santiago, Chile (Biographisches Lexikon, 1962; Deutsche Biographische Enzyklopa¨die, 1997; O¨sterreichisches Biographisches Lexikon, 1969; Anonymous, 1932). Thus, Rudolf Kraus and his co-workers E. Prˇ ibram and A. Prantschoff found that the El Tor vibrios coincided in cell morphology and biochemical characteristics with that of cholera vibrios, ‘‘but they possess additional peculiarities differing from cholera vibrio Koch; they all produce haemotoxins (haemolysins), and an acute acting toxin’’. ‘‘Therefore an exceptional position may be due to these vibrios’’ [Deshalb mag diesen Vibrionen eine Sonderstellung zukommen]. However, they found that haemolysin and toxin production was not restricted to El Tor strains, but could be detected in other choera-like vibrios as well (including those listed in Table 1). The haemotoxins showed cross reaction with each of the anti-haemotoxic antisera (Kraus and Prantschoff, 1906). The authors demonstrated the serological cross reaction with anti-cholera and anti-El Tor cholera antisera: ‘‘High-grade cholera antiserum agglutinate El Tor vibrios in the same manner as cholera vibrios. It was shown furthermore, that antiEl Tor antisera agglutinate with cholera strains with the same high titres as El Tor strains y With these results in mind we should suppose these 6 strains to be cholera vibrios’’. The titres of agglutination were higher with anticholera sera than the El Tor antisera but nevertheless ‘‘yeven these differences are incapable to shake the presently accepted specificity of agglutinating cholera serum.’’ Soluble haemotoxin production was problematic. In an earlier paper Kraus (1903) was unable to detect the
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production of a soluble haemotoxin by cholera vibrios in contrast to the cholera-like Vibrio Nasik (the socalled ‘‘Vibrio Nasik’’ was obtained through Prof. Paltauf, Vienna, who had received the strain from Alexandre Marmorek at the Institute Pasteur Paris, with the designation ‘‘cholera vibrio’’. Vibrio Nasik had originally been isolated from a human case of cholera nostras in India). Kolle and Meinicke (1905) also failed to detect any soluble haemotoxin production by El Tor vibrios. In contrast, Prˇ ibram (1907) and Meinicke (1905) were able to detect soluble haemotoxin and so did Kraus and Prˇ ibram (1906) upon re-examination. The haemotoxin could be detected in 2-day-old broth cultures of the El Tor vibrios using rabbit erythrocytes with haemolysis occurring after 1 hour. In the same way, a sterile culture filtrate was haemolytic but heating to 58 1C destroyed the haemolysin (Prˇ ibram, 1907). On sheep- and goat blood agar plates clear halos were seen around the El Tor vibrio colonies while V. cholerae did not change the blood agar. After immunisation of rabbits and goats with broth cultures of El Tor vibrios the antisera neutralised the haemolysins of all strains in dilutions up to 1:1000. It was also found that the antihaemolysin against El Tor vibrios neutralised the haemolysin of the so-called Vibrio Nasik and vice versa (Kraus and Prˇ ibram, 1906; Kraus and Prantschoff, 1906). Both antisera inhibited also the haemolytic activity of many other cholera-like bacteria, a result obtained previously by Meinicke (1905). Kraus, with Prˇ ibram and Prantschoff detected another feature peculiar to El Tor vibrios and not Koch’s cholera vibrio: ‘‘ybroth cultures of cholera vibrios do not produce a soluble toxin.yin the sense of diphtheriaor tetanus toxin’’ whereas the six El Tor vibrios did. ‘‘After intravenous injection of two-day-old broth cultures rabbits y died within a few minutes up to 1 h. y The rabbits breathed more frequently, became restless, developed pareses and paralyses, and ruined after some terminal convulsions y The toxicity could be observed also after the application of filtrates’’. The same poison was found in cultures of ‘‘V. Nasik’’ (which according to Rothberger (1905) was cardiotoxic), ‘‘but not in cultures of cholera or cholera-like vibrios like V. Metschnikoff, V. Finkler-Prior or V. Danubicus’’. Kraus and Prˇ ibram concluded: ‘‘By these studies new characteristics of El Tor vibrios were detected which were never found until now in cholera vibrios isolated from typical cases of cholera. It must be decided whether or not we are justified to designate these strains as cholera strains and to identify them with cholera vibrio Koch’’. Kraus and Prˇ ibram had no difficulties in preparing an antitoxin against this ‘‘active acting toxin’’. First they found that normal serum from horses or goats had neutralising activity only after incubation for 1=2 h at 37 1C before injection into the experimental animals. In
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contrast, antitoxic antiserum against the toxin of El Tor vibrios was able to ‘‘paralyse’’ ( ¼ neutralise) immediately the activity of El Tor toxin. An antiserum obtained by immunisation with the toxin of V. Nasik was able to neutralise the toxin of El Tor vibrios. The paper of Kraus and Prantschoff (1906) is concerned with the production of haemotoxins and toxins in the 32 cholera-like vibrios isolated by F. Gotschlich (1906). Most of the strains produced haemotoxin in two-day-old broth cultures, all of which did so after 5 days. Antihaemolysins raised against the El Tor vibrios neutralised the haemotoxins of all vibrios, El Tor as well as cholera-like vibrios. Likewise, antihaemolysins of the cholera-like vibrios neutralised the haemolysins of all vibrios. Financial restraints meant the production of the acute acting toxin could only be examined in a few of the 32 cholera-like vibrios. The result was the same as for the haemotoxins. All of them secreted the soluble acute acting toxin and antisera showed cross-neutralisation. Thus, Kraus and his coauthors grant an exceptional position to the El Tor vibrios. In common with V. cholerae they shared morphological and cultural characteristics, as well as agglutination by cholera antiserum and a positive Pfeiffer reaction. They differed by the production of haemolysin and soluble toxin. Later Kraus (1909) had re-examined the El Tor vibrios and declared that the strains haemolytic on sheep blood agar plates were not responsible for epidemic cholera. He called them ‘paracholera vibrios’ and believed that they were able to cause only sporadic cases of a cholera-like disease. The distinction between the haemolytic and haemodigestive activity of the El Tor vibrio is recalled in detail by Pollitzer (1959) who recognises the important contributions of Schumacher and van Loghem using plates with whole blood and lysed blood. El Tor showing clear zones of clearing on the lysed blood, but not V. cholerae. If haemolysis in vibrios caused uncertainty in studies between the V. cholerae biotypes one hundred years ago, then they continue to intrigue workers today. The identity of the El Tor haemolysin has since been shown to be a pore-forming toxin (HlyA) but a further haemolysin present in O1 V. cholerae has been identified (discussed in (Zhang and Austin, 2005)). In failing to identify a soluble toxin in V. cholerae Kraus confirmed earlier work of Richard Pfeiffer (1894, 1895a, b) but the evidence of an additional toxin gave the El Tor vibrios equal if not greater significance than Koch’s comma vibrio. However, many years elapsed before it became clear that Felix Gotschlich’s El Tor vibrio is a biotype of V. cholerae, distinct from the classical biotype with the general scientific perception being that El Tor vibrios were not as virulent as Koch’s V. cholerae. The delay in realisation that El Tor strains were toxigenic and able to cause epidemic cholera meant that International Sanitary Regulations resulting from
the 15th World Health Assembly meeting only recognised El Tor strains as true cholera in 1962, triggered by the start of the 7th cholera pandemic in Indonesia in 1961 (Kamal, 1974). The exhaustive treatise on cholera by Pollitzer in 1959 typified the viewpoint of many that El Tor strains were non-pathogenic as late as 1959. [Intruigingly, Kraus in 1909 had proposed that the El Tor strains were responsible for epidemic cholera (Kraus, 1909).] The early studies on the acute soluble toxin reported by Kraus are difficult to interpret because of contaminating endotoxin. The toxin was considered for many years to be intracellular and only released upon lysis of the bacterial cell (a problem in common with studies of Shigella toxins of the period as well). Nevertheless, it remains sobering that the elucidation of the mode of action of cholera toxin took another 50 years. Whilst the name El Tor is perpetuated alongside cholera Gotschlich, Kraus and Ruffer all considered a role for the organism in dysentery. Dysenteric disease was a far more frequent cause of morbidity and mortality than cholera at the El Tor quarantine camp. Table 2 shows how infrequent cholera outbreaks actually were in Egypt. Having drawn the work of Gotschlich and others to one’s attention, can we confidently explain how Vibrio cholerae El Tor strains were isolated from people without cholera? Our understanding of the sequential series of phage infections, of which one codes for the classic cholera toxin provides a theoretical framework around which we can now understand the isolation of non-toxigenic Vibrio cholerae-like organisms from rivers (Faruque and Mekalanos, 2003). The carrier state had been identified by 1906 and it remains the case that V. cholerae rarely adopts a long lived carrier state.
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Bujwid, O., 1888. Neue Methode zum Diagnosticiren und Isoliren der Cholerabakterien. Cbl. Bakteriol. Parasitenkde. 4, 494–496. Bulloch, W., 1938. The History of Bacteriology. Oxford Univ. Press, London. Dunbar, W.P., 1893. Untersuchungen u¨ber choleraa¨hnliche Wasserbacterien. Dtsch. Med. Wochenschr. 19, 799–800. Eyler, J.M., 2001. Changing assessments of John Snow’s and William Farr’s cholera studies. Soz.-Pra¨ventivmed. 46, 225–262. Faruque, S.M., Mekalanos, J.J., 2003. Pathogenicity islands and phages in Vibrio cholerae evolution. Trends Microbiol. 11, 505–510. Finkler, D., Prior, J., 1884. U¨ber den Bacillus der Cholera nostras und seine Cultur. Dtsch. Med. Wochenschr. 10, 632–634. Gamaleia, M.N., 1888. Vibrio metschnikovi [sic!] (n. sp.) et ses rapports avec le microbe du chole´ra asiatique. Ann. Inst. Pasteur 2, 482–488. Gotschlich, E., 1904. Allgemeine Prophylaxe der Infektionskrankheiten. In: Kolle, W., Wassermann, A. (Eds.), Handbuch der Pathogenen Mikroorganismen, vol. 4/1. Gustav Fischer, Jena, pp. 1–65. Gotschlich, F., 1906. U¨ber cholera- und choleraa¨hnliche Vibrionen unter den aus Mekka zuru¨ckkehrenden Pilgern. Z. Hyg. Infektionskrankh. 53, 281–304. Gu¨nther, C., 1893. Weitere Studien u¨ber den Vibrio Berolinensis. Arch. Hyg. 19, 214–222. Howard-Jones, N., 1975. The scientific background to the International Sanitary Conferences 1851–1938. World Health Organisation, Geneva. Howard-Jones, N., 1984. Robert Koch and the cholera vibrio: a centenary. Brit. Med. J. i, 379–381. India Office, Great Britain, 1885. Cholera: Inquiry by Doctors Klein and Gibbes, and Transactions of a Committee Convened by the Secretary of State for India in Council. India Office, London. Iva´noff, M., 1893. Ueber eine neue choleraa¨hnliche Vibrionenart. Z. Hyg. Infectionskrankh. 14, 134–138. Judicial Commission, 1965. Int. Committee on Bacteriol. Nomenclature. Opinion. Int. Bull. Bacteriol. Nomenclature 15, 185. Kamal, A.M., 1974. The seventh pandemic of cholera. In: Barua, D., Burrows, W. (Eds.), Cholera. W.B. Saunders & Co., Philadelphia, PA, pp. 1–14. Kaufmann, P., 1892. Die Quaranta¨nestation El Tor: Beobachtungen wa¨hrend eines 35-ta¨gigen Aufenthaltes daselbst. A. Hirschwald, Berlin. Koch, R., 1883. Der Seitens des Geh. Reg. Rath Dr. R. Koch an den Staatssecreta¨r des Innern, Herrn Staatsminister v. Boetticher, Exzellenz, erstattete Bericht. Dtsch. Med. Wochenschr. 9, 615–617. Koch, R., 1884a. Fu¨nfter Bericht des Leiters der deutschen wissenschaftlichen Commission zur Erforschung der Cholera, Geheimen Regierungsrathes Dr. Koch (7. Januar 1884). Dtsch. Med. Wochenschr. 10, 111–112. Koch, R., 1884b. Sechster Bericht der deutschen wissenschaftlichen Commission zur Erforschung der Cholera, Geh. Regierungsrathes Dr. Koch (2. Februar 1884). Dtsch. Med. Wochenschr. 10, 191–192.
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Koch, R., 1884c. VII. Bericht des Leiters der deutschen wissenschaftlichen Commission zur Erforschung der Cholera, Geh. Regierungsrathes Dr. Koch, aus Kalkutta, 4. Ma¨rz. Dtsch. Med. Wochenschr. 10, 221–222. Ko¨hler, W., 1997. Die Entdeckung der Pra¨zipitationsreaktion durch Rudolf Kraus. Immun. Infekt. 2, 77–80. Kolle, W., 1903. Cholera asiatica. In: Kolle, W., Wassermann, A. (Eds.), Handbuch der Pathogenen Mikroorganismen, vol. 3. Gustav Fischer, Jena, pp. 1–74. Kolle, W., Gotschlich, E., 1903. Untersuchungen u¨ber die bakteriologische Choleradiagnostik und Specifita¨t des Koch’schen Choleravibrio. Z. Hyg. Infektionskrankh. 44, 1–128. Kolle, W., Meinicke, E.M., 1905. Bull. quarant. hebd. publie´ par le conseil sanit. marit. et quarant. d’Egypt, No 276. Kraus, R., 1903. Ueber ein akut wirkendes Bakterientoxin. Cbl. Bakteriol. Parasitenkde. Infektionskrankh. 1. Abt. Orig. 34, 488–496. Kraus, R., 1909. Ueber den derzeitigen Stand der a¨tiologischen Diagnose und der antitoxischen Therapie der Cholera asiatica. Wien. Klin. Wochenschr. 22, 43–47. Kraus, E., Prantschoff, A., 1906. Ueber Choleravibrionen und andere Vibrionen. III. Ueber Identita¨t der Ha¨motoxine und der Toxine, der Vibrionen sowie deren Antitoxine. Cbl. Bakteriol. Parasitenkde. Infektionskrankh. 1. Abt. Orig. 41, 377–379 480–487.1. Kraus, R., Prˇ ibram, E., 1906. Ueber Choleravibrionen und andere pathogene Vibrionen. I. Ueber die Beziehungen der Vibrionen El Tor zu dem Choleravibrio. Cbl. Bakteriol. Parasitenkde. Infektionskrankh. 1. Abt. Orig. 41, 15–21, 155–161. Meinicke, E.M., 1905. Ueber die Ha¨molysine der choleraa¨hnlichen Vibrionen. Z. Hyg. Infectionskrankh. 50, 165–185. Neisser, M., 1893. Ueber einen neuen Wasser-Vibrio, der die Nitrosoindol-Reaction liefert. Arch. Hyg. 19, 194–213. Ogawa, M., 2000. Uneasy bedfellows: science and politics in the refutation of Koch’s bacterial theory of cholera. Bull. Hist. Med. 74, 671–707. Pacini, F., 1854. Osservazioni Microscopiche e Deduzioni Patologiche Sul Cholera Asiatica. Bencini, Firenze. Pasquale, A., 1891. Ricerche batteriologiche sul colera a Massaua e considerazione igieniche. Giorn. Med. Milit. 39, 1009–1031. Petri, R.J., 1893. Der Cholerakurs am Kaiserlichen Gesundheitsamte. Richard Schoetz, Berlin. Pfeiffer, R., 1894. Studien zur Choleraa¨tiologie. Z. Hyg. Infectionskrankh. 16, 268–286. Pfeiffer, R., 1895a. Die Differentialdiagnose der Vibrionen der Cholera asiatica mit Hu¨lfe der Immunisirung. Z. Hyg. Infectionskrankh. 19, 75–100. Pfeiffer, R., 1895b. Weitere Mittheilungen u¨ber die specifischen Antiko¨rper der Cholera. Z. Hyg. Infectionskrankh. 20, 198–219. 1 There seems to be a mistake in numbering the articles: The papers by Kraus and Prˇ ibram were denoted as ‘‘I’’ and appeared in two parts in the issues 1 and 2 of volume 41; the first part of Kraus and Prantschoff’s paper was designated as ‘‘III’’ (should it be II?), appeared in issue 3, and the ‘‘end’’ of this paper, designated also as ‘‘III’’ in issue 4 of vol. 41.
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Pfeiffer, R., 1896. Die Spirillen. In: Flu¨gge, C. (Ed.), Die Mikroorganismen, third ed., vol. II. F.C.W. Vogel, Leipzig, pp. 527–599. Pollitzer, R., 1959. Cholera. World Health Organisation Monograph Series 43, Geneva. Prˇ ibram, E., 1907. U¨ber Bakterienha¨motoxine (Lysine) und Antiha¨motoxine. In: Kolle, W., Wassermann, A. (Eds.), Handbuch der Pathogenen Mikroorganismen. Erg.-Bd. I. Gustav Fischer, Jena, pp. 291–346. Rimpau, W., 1935. Die Entstehung von Pettenkofers Bodentheorie und die Mu¨nchner Choleraepidemie vom Jahre 1854. Richard Schoetz, Berlin. Rothberger, C.J., 1905. Ueber ein akut wirkendes Bakterientoxin. Cbl. Bakteriol. Parasitenkde. Infektionskrankh. 1. Abt. Orig. 38, 165–172. Ruffer, M.A., 1899. Measures taken at Tor and Suez against ships coming from the Red Sea and the Far East. Lancet ii, 1801–1806. Ruffer, M.A., 1907. Researches on the bacteriological diagnosis of cholera. Brit. Med. J. i, 735–742. Santifaller, L., Obermeyer, E. (Eds.), 1969. O¨sterreichisches Biographisches Lexikon 1850–1951. H. Bo¨hlau Nachf., Wien, Ko¨ln, Graz.
Saur, K.G., 1997. Deutsche Biographische Enzyklopa¨die (DBE). Mu¨nchen. Sanderson, J.B., 1885. The cholera and the comma bacillus. Brit. Med. J. i, 1076–1077. Shousha, A.T., 1947. Cholera epidemic in Egypt (1947). Bull. World Health Org. 1, 353–381. Snow, J., 1849/1855. On the Mode of Communication of Cholera. London (31pp.; second ed. 1855, 162pp.). Uhlenhuth, P., 1930. Emil Gotschlich zum 60. Geburtstage. Zbl. Bakteriol. Parasitenkde. Infektionskrankh. 1. Abt. Orig. 116, I–IV. von Pettenkofer, M., 1887. Zum gegenwa¨rtigen Stand der Cholerafrage. Oldenbourg, Mu¨nchen. Zhang, X.-H., Austin, B., 2005. Haemolysins in Vibrio species. J. Appl. Microbiol. 98, 1011–1019.
Werner Ko¨hler Adolf-Reichwein-Str. 26, D-07745 Jena, Germany Simon P. Hardy Norwegian Veterinary School, PO Box 8146, N-0033 Oslo, Norway
International Journal of Medical Microbiology 296 (2006) 333–340 www.elsevier.de/ijmm
EDITORIAL
Zentralblatt fu¨r Bakteriologie – 100 years ago: Early considerations of the El Tor vibrios The most famous advocate of the water borne transmission of cholera was John Snow (1849/1855). Already famous for being the person to introduce ether for anaesthesia in England, notably to Queen Mary (Bulloch, 1938), Snow’s work as an epidemiologist still commands attention from scholars (e.g. Eyler, 2001). In the mid nineteenth century the miasmatic theory of cholera was prevalent and editors of scientific journals were reluctant to publish his findings on the water-borne origin of the disease, so he did it at his own cost (Snow, 1849/1855). The water-borne epidemiology of cholera was confirmed by Robert Koch and the members of the ‘‘German Cholera Expedition’’ (without mentioning John Snow) when they found in 1884 the vibrios in the so-called ‘‘tanks’’ in Bengal. Tanks are water-basins (small lakes or artificially basins), surrounded by huts, and served for water-storage. The inhabitants used the water not only as sources of drinking-water, but also for bathing and washing clothes. In the small epidemic observed by Koch even the soiled clothes of the first cholera victim were washed in this peculiar tank (Koch, 1884c). Koch’s verdict, that only diseased humans or carriers were the source of new cases of cholera was one of many epochal insights but over time has had to be rethought concerning cholera in light of the movement of V. cholerae between estuarine habitats and man. Koch announced in his report from Alexandria dated 17th of September 1883, that ‘‘the mission found the constant presence of a specific bacillus in the intestinal of persons dying from cholera’’ (Koch, 1883). He concluded ‘‘that there could be no doubt that the bacillus stood in some relation to the cholera process’’ (Howard-Jones, 1984), but he had not obtained a pure culture. The isolation was announced in his fifth dispatch dated 7th of January 1884 (Koch, 1884a), and this date should be accepted as the real time of the discovery of the aetiological agent of cholera. The term ‘‘comma bacillus’’ was coined in his 6th dispatch, dated 2nd of February, when he described the germ as ‘‘a little 1438-4221/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2006.04.001
bent, like a comma’’ (‘‘ein wenig gekru¨mmt, einem Komma a¨hnlich’’) (Koch, 1884b). What Koch apparently had not seen or had ignored was a booklet published in 1854 by Filippo Pacini (1812–1883), Professor of Anatomy at Florence. There Pacini described what he had seen in cholera dejections: ‘‘miriardi di vibrioni’’ (Pacini, 1854). Coming to the conclusion that these vibrios were the causative agents, Pacini’s work was honoured by the Judicial Commission of the International Committee on Bacteriological Nomenclature, 1965, when they adopted ‘‘Vibrio cholerae Pacini, 1854’’ as the official, correct name of the cholera vibrio. Questions over the validity of Koch’s findings appeared shortly after Koch’s announcement (see von Pettenkofer, 1887; Rimpau, 1935; Anonymous, 1884a, b; India Office, 1885; Sanderson, 1885) and recent work has reconsidered the circumstances surrounding the criticisms of Koch’s work by Klein and the British Commission (Ogawa, 2000). Remarkably, the scepticism over V. cholerae as the cause of cholera persisted for almost 20 years (Howard-Jones, 1975). In part this was due to the ‘‘yseveral hundreds different species of vibrios y isolated from water, manure, putrid fluids, human dejections etc y. which are more or less similar to cholera vibrios’’ in their cultural and biochemical activities (Kolle, 1903). Examples of which are listed in Table 1. Other reasons included the problems of reproducing the disease in animal models. In order to reduce confusion in the identification of cholera-like vibrios and V. cholerae, Robert Koch, Martin Kirchner and Wilhelm Kolle wrote a pamphlet on the bacteriological diagnosis of cholera which was published in 1902 as: ‘‘Writ by the Minister of religious, educational and medical affairs, regarding instruction for the bacteriological ascertainment of cases of cholera, from 6th of November 1902’’ (‘‘Erlaß des Ministers der geistlichen, Unterrichts- und Medizinalangelegenheiten, betreffend Anleitung fu¨r die bakteriologische Feststellung der Cholerafa¨lle, vom 6. November 1902’’). The following examinations must be done in cases of
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Table 1.
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Selected Vibrio species similar to V. cholerae isolated from a variety of sources prior to 1900
Designation
Reference(s)
Isolated from
Possible species (Bergey’s Manual, 1974)a
V. V. V. V. V. V. V.
Finkler and Prior, 1884 Gamaleia, 1888 Pasquale, 1891 Pasquale, 1891 Dunbar, 1893 Iva´noff, 1893 Neisser, 1893; Gu¨nther 1893
Cholera nostras (man) Gastroenteritis in poultry Water well in Ghinda near Massaua Diarrhoea (man) River Elbe Faeces of man with typhoid fever Tap-water from the River Spree
V. cholerae biotype proteus V. cholerae biotype proteus V. choleraeb Unknown V. cholerae biotype albensisc V. cholerae varietyd V. cholerae (low virulence)? Contamination ?e
finkleri metschnikovii Ghinda Massauah phosphorescens Iwanoff Berolinensis
a
In Bergey’s Manual of Systematic Bacteriology, 1st edit., 1984, vol. 1 these ‘‘species’’ were no longer included. Not mentioned in Bergey’s. c According to Bergey’s Manual (1974) the type strain ATCC 14547 is indistinguishable from V. cholerae by DNA homology. d Bergey’s 6th ed., 1948. e Pfeiffer (1896) believed that the vibrios were not present in the stools of the patient but an external contamination. b
suspected cholera: Microscopical examination of stool (smears from flakes of the dejecta); 2. Alkaline gelatine plates; 3. Alkaline agar plates; 4. Enrichment in alkaline peptone-water; 5. Examination of pure cultures by agglutination, and 6. Pfeiffer’s reaction, i.e. serum killing of the vibrios in immune animals (Kolle, 1903). The document was also translated and published in England (Note the absence of the’’cholera-red reaction’’ (nitroso-indole reaction), introduced by Poehl in 1886 (cited by Petri, 1893) and proposed independently by Bujwid (1887, 1888) as a marker of V. cholerae). This spectrum of mostly non-specific methods helps explain why the laboratory diagnosis of cholera was so confused. Given this history of uncertainty as to the significance of the cholera-like vibrios the situation was muddied further by periodic but repeated isolation of yet more varieties of vibrio-like bacteria from pilgrims returning from Mecca. At the turn of the century (1900) there were two main routes of the pilgrimage to Mecca (also known as the Haj): pilgrims from Asia Minor, mostly Turks, landed at Yenbo before travelling by foot or camel to Medina and then Mecca (Fig. 1). The second route taken by most other nationalities (Egyptians, Algerians, Syrians, and Russians) arrived at the port of Jiddah and then firstly to Mecca. The returning pilgrims were forced to go via the quarantine camps, the closest camp under ‘‘Egyptian’’ control (rather than Turkish) was El Tor. The time taken to reach El Tor from Jiddah and Yenbo was between three to eight weeks effectively preventing any cholera entering Europe via the Suez Canal. When transportation by boat was established the time taken was reduced considerably with consequent increase in the number of pilgrims still excreting vibrios. The dreadful overcrowded conditions onboard have been described by Ruffer (1899) and make for harrowing reading. The sanitary station at El Tor was located at the West Coast of the Sinai Peninsula in ‘‘a spot of unsurpassed
attractiveness’’ (Anonymous, 1907), and was established in 1877 following discussions of the first International Sanitary Conferences at Constantinople in 1866. Several bacteriology laboratories were established at the quarantine stations, not just at El Tor but also at Alexandria, Suez and Port Said in order to investigate the pilgrims. Across the Red Sea there were further quarantine stations in the Turkish occupied region (now Saudi Arabia; Anonymous, 1907). The pilgrims had to pass through the quarantine station in numbers reaching 20,000 people at a time. The camp at El Tor was surrounded by wire-fencing ensuring that after disembarkation there was only one way to the camp – through the disinfection establishment (‘‘Desinfektionsanstalt’’). The pilgrims were made to strip, female European physicians and nurses took care for the female pilgrims, and they had to douche. Meanwhile the clothes and the personal effects were disinfected by steam or chemical methods. Then the pilgrims were sent to ‘‘sections’’ again fenced off by barbed wire (Fig. 2). If infectious diseases broke out they were restricted to this area. The length of the quarantine depended upon the epidemiological situation in the Hedjaz region. If there was no cholera or plague (‘‘pe´le´rinage net’’) the duration was three days, in the case of epidemics in the area (‘‘pe´le´rinage brut’’) 18 days (Gotschlich, 1904; Kaufmann, 1892). Immediately upon arriving in Alexandria (Sir) Marc Armand Ruffer (1859–1917), President of the Sanitary, Maritime and Quarantine council of Egypt, based at the Port Vieux Laboratory, Alexandria, Egypt, orchestrated investigations into the bacteriology of cholera and dysentery. As early as 1897 Dr. Galvani isolated a vibrio from a dead pilgrim which agglutinated with cholera antiserum (Ruffer, 1907; Bryceson, 1977). Subsequent investigations of the intestinal bacteriology of the pilgrims who had died at El Tor quarantine camp revealed a further five strains isolated from pilgrims who had been suffering with dysentery and not cholera.
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Fig. 1. Map showing the location of El Tor at the Red Sea and of other towns in the Hedjaz area.
Fig. 2. The quarantine camp at El Tor as drawn by Paul Kaufmann (1892). The sketches give no indication as to the conditions of the camp when housing 20,000 pilgrims.
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Table 2.
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Years in which cholera was recorded in Egypta
Year of epidemic/ outbreak
World Pandemic
Number of deaths
1831 1834 1837 1848 1850 1857 1865
2nd 2nd 2nd 3rd 3rd 3rd 4th
1882/1883 1895/1896 1902 1947
5th 5th 6th 7th
150,000 Similar to above ? ? ? ? 460,000 (30,000 pilgrims) 48,000–58,000 16,571–37,000 34,600 20,800
a
Compiled from (Shousha, 1947; Anonymous, 1951; Pollitzer, 1959). Ranges represent varying values in the different sources.
Cholera had not been reported that year at the Haj and did not appear there until 1902 (see Table 2). In 1903 Kolle and Gotschlich examined many other cholera-like vibrios including two strains obtained from Heinrich Bitter who had isolated these (and other strains) from pilgrims with and without cholera at El Tor (Kolle and Gotschlich, 1903). Heinrich Bitter (1863–1918) studied medicine in Munich, where he graduated MD in 1886. After a short time at the Clinic of Dermatology at the University of Wu¨rzburg, he became assistant at the Institute of Hygiene at Breslau (habilitation 1891). In 1893 he was offered the post as an ‘Inspector of Sanitary Matters’’ (Inspektor des Sanita¨tswesens) at Cairo, where he built a new Institute of Hygiene. Robert Koch asked Carl Flu¨gge for a successor for Heinrich Bitter who recommended Emil Gotschlich (1870–1949). Thus, Gotschlich became head of the Sanitary Board at Alexandria in 1896 as well as Egyptian delegate at the international ‘‘Conseil Sanitaire, Maritime et Quarantinaire’’. Emil Gotschlich had trained under Carl Flu¨gge at the Institute of Hygiene, University of Breslau. After two years there he accepted the invitation to Egypt. He lost his post at the beginning of World War I, but later became director of the Institutes of Hygiene at Halle, Saarbru¨cken, Giessen and Heidelberg (Uhlenhuth, 1930). By 1905 Emil’s younger brother Felix Gotschlich was working at the laboratories of the ‘‘Conseil sanitaire maritime et quarantenaire d’Egypt’’ in Alexandria and continuing the work on the role of cholera-like vibrios in enteric disease. Felix Gotschlich found further varieties of the comma bacillus in the intestines at autopsy from patients at the sanitation camp at El Tor. The dead men had arrived at El Tor in different ships and had died
1–13 days after disembarkation. They had not suffered from cholera (which had not been reported at the festival that year) but instead had dysenteric disease (which had been reported). Felix Gotschlich isolated 38 strains of vibrio from 6 people who had died of colitis, colitis gangraenosa or dysentery at El Tor (and two further strains the following year, 1906, under similar circumstances). Six strains were agglutinated by cholera antisera (Gotschlich, 1906). In common with Koch’s Vibrio cholerae they were positive for Pfeiffer’s reaction, had typical morphology of cholera vibrios (one flagellum), liquefied gelatine, coagulated milk, were pathogenic for guinea pigs and apathogenic for pigeons. The cholera-red reaction was positive with the exception of one strain. The latter one showed a positive reaction too after addition of some potassium nitrate to the peptone-water medium. Gotschlich did not mention the behaviour on blood agar plates. In contrast the other 32 strains of vibrios not agglutinated by cholera antiserum all showed haemolysis, but presented varying cholera-red reactions and milk coagulation. All were pathogenic for guinea pigs, a few for pigeons also. Gotschlich concluded that the six vibrios (agglutinated by cholera antisera) were ‘‘real cholera vibrios’’, and that the diseased pilgrims were ‘‘cholera carriers’’. He gave no special name to these vibrios since he was sure about their identity as V. cholerae. Gotschlich (1906) also reported that his brother Emil had examined a vibrio isolated by Ruffer in 1897 in El Tor from faeces of a pilgrim without any signs of cholera. This strain was morphologically identical with V. cholerae, agglutinated by cholera antiserum, highly pathogenic for guinea pigs, showed a positive cholera-red reaction but was negative in Pfeiffer’s reaction. This is likely to be the same organism that Ruffer credited to Dr. Galvani. Reviewing the bacteriology of Gotschlich and Dr. Zirolia, Director of the Port Said laboratory, Ruffer (1907) highlighted the uncertainty over the variation in number of flagella and, more importantly, the unreliable bacteriological diagnosis obtained by using anticholera serum since the El Tor strains reacted. As the isolates were from cases of dysentery Ruffer felt that the use of anti-cholera serum was insufficient. The vibrios were being forwarded to European laboratories on a regular basis and the vibrios studied by Felix Gotschlich were sent to the State Serotherapeutic Institute in Vienna. There, Rudolf Kraus (1868–1932) carried out further examinations of the organisms and 100 years ago in this journal Kraus, published his findings in two papers, assisted by E. Prˇ ibram and A. Prantschoff (Kraus and Prˇ ibram, 1906; Kraus and Prantschoff, 1906). Rudolf Kraus was born on 31st of October 1868 in Jungbunzlau/Bohemia (now Mlada´ Boleslaw), studied medicine in Prague and graduated there in 1893. He
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then became assistant at the Institute of Pathology in Vienna, trained under Richard Paltauf (director of the Institute of Pathology as well as of the Serotherapeutic Institute). In 1903 he habilitated in the field of general and experimental pathology, and was appointed in 1910 as Assistant Professor. In 1913 he became director of the Bacteriological Institute at Buenos Aires, later of the Serological Institute at Butantan (Sa˜o Paulo). Ten years later he came back to Vienna and succeeded Paltauf as a director of the Serotherapeutic Institute. In 1928 he was invited by the Chilean government to establish a new ‘‘Istituto Bacteriologico de Chile’’ in Santiago de Chile also becoming director general of the International Society of Microbiology in 1930. Kraus is known to microbiologists by the detection of bacterial precipitation (Ko¨hler, 1997), as well as editor of several important textbooks: the 3rd edition of ‘‘Handbuch der pathogenen Mikroorganismen’’ 1927 (together with Kolle and Uhlenhuth), ‘‘Handbuch der Immunita¨tsforschung und experimentellen Therapie’’ (together with C. Levaditi, 2nd ed. 1914), and ‘‘Handbuch der mikroskopischen Technik’’ (together with P. Uhlenhuth 1922–24). Rudolf Kraus died on 15th/16th July 1932 in Santiago, Chile (Biographisches Lexikon, 1962; Deutsche Biographische Enzyklopa¨die, 1997; O¨sterreichisches Biographisches Lexikon, 1969; Anonymous, 1932). Thus, Rudolf Kraus and his co-workers E. Prˇ ibram and A. Prantschoff found that the El Tor vibrios coincided in cell morphology and biochemical characteristics with that of cholera vibrios, ‘‘but they possess additional peculiarities differing from cholera vibrio Koch; they all produce haemotoxins (haemolysins), and an acute acting toxin’’. ‘‘Therefore an exceptional position may be due to these vibrios’’ [Deshalb mag diesen Vibrionen eine Sonderstellung zukommen]. However, they found that haemolysin and toxin production was not restricted to El Tor strains, but could be detected in other choera-like vibrios as well (including those listed in Table 1). The haemotoxins showed cross reaction with each of the anti-haemotoxic antisera (Kraus and Prantschoff, 1906). The authors demonstrated the serological cross reaction with anti-cholera and anti-El Tor cholera antisera: ‘‘High-grade cholera antiserum agglutinate El Tor vibrios in the same manner as cholera vibrios. It was shown furthermore, that antiEl Tor antisera agglutinate with cholera strains with the same high titres as El Tor strains y With these results in mind we should suppose these 6 strains to be cholera vibrios’’. The titres of agglutination were higher with anticholera sera than the El Tor antisera but nevertheless ‘‘yeven these differences are incapable to shake the presently accepted specificity of agglutinating cholera serum.’’ Soluble haemotoxin production was problematic. In an earlier paper Kraus (1903) was unable to detect the
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production of a soluble haemotoxin by cholera vibrios in contrast to the cholera-like Vibrio Nasik (the socalled ‘‘Vibrio Nasik’’ was obtained through Prof. Paltauf, Vienna, who had received the strain from Alexandre Marmorek at the Institute Pasteur Paris, with the designation ‘‘cholera vibrio’’. Vibrio Nasik had originally been isolated from a human case of cholera nostras in India). Kolle and Meinicke (1905) also failed to detect any soluble haemotoxin production by El Tor vibrios. In contrast, Prˇ ibram (1907) and Meinicke (1905) were able to detect soluble haemotoxin and so did Kraus and Prˇ ibram (1906) upon re-examination. The haemotoxin could be detected in 2-day-old broth cultures of the El Tor vibrios using rabbit erythrocytes with haemolysis occurring after 1 hour. In the same way, a sterile culture filtrate was haemolytic but heating to 58 1C destroyed the haemolysin (Prˇ ibram, 1907). On sheep- and goat blood agar plates clear halos were seen around the El Tor vibrio colonies while V. cholerae did not change the blood agar. After immunisation of rabbits and goats with broth cultures of El Tor vibrios the antisera neutralised the haemolysins of all strains in dilutions up to 1:1000. It was also found that the antihaemolysin against El Tor vibrios neutralised the haemolysin of the so-called Vibrio Nasik and vice versa (Kraus and Prˇ ibram, 1906; Kraus and Prantschoff, 1906). Both antisera inhibited also the haemolytic activity of many other cholera-like bacteria, a result obtained previously by Meinicke (1905). Kraus, with Prˇ ibram and Prantschoff detected another feature peculiar to El Tor vibrios and not Koch’s cholera vibrio: ‘‘ybroth cultures of cholera vibrios do not produce a soluble toxin.yin the sense of diphtheriaor tetanus toxin’’ whereas the six El Tor vibrios did. ‘‘After intravenous injection of two-day-old broth cultures rabbits y died within a few minutes up to 1 h. y The rabbits breathed more frequently, became restless, developed pareses and paralyses, and ruined after some terminal convulsions y The toxicity could be observed also after the application of filtrates’’. The same poison was found in cultures of ‘‘V. Nasik’’ (which according to Rothberger (1905) was cardiotoxic), ‘‘but not in cultures of cholera or cholera-like vibrios like V. Metschnikoff, V. Finkler-Prior or V. Danubicus’’. Kraus and Prˇ ibram concluded: ‘‘By these studies new characteristics of El Tor vibrios were detected which were never found until now in cholera vibrios isolated from typical cases of cholera. It must be decided whether or not we are justified to designate these strains as cholera strains and to identify them with cholera vibrio Koch’’. Kraus and Prˇ ibram had no difficulties in preparing an antitoxin against this ‘‘active acting toxin’’. First they found that normal serum from horses or goats had neutralising activity only after incubation for 1=2 h at 37 1C before injection into the experimental animals. In
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contrast, antitoxic antiserum against the toxin of El Tor vibrios was able to ‘‘paralyse’’ ( ¼ neutralise) immediately the activity of El Tor toxin. An antiserum obtained by immunisation with the toxin of V. Nasik was able to neutralise the toxin of El Tor vibrios. The paper of Kraus and Prantschoff (1906) is concerned with the production of haemotoxins and toxins in the 32 cholera-like vibrios isolated by F. Gotschlich (1906). Most of the strains produced haemotoxin in two-day-old broth cultures, all of which did so after 5 days. Antihaemolysins raised against the El Tor vibrios neutralised the haemotoxins of all vibrios, El Tor as well as cholera-like vibrios. Likewise, antihaemolysins of the cholera-like vibrios neutralised the haemolysins of all vibrios. Financial restraints meant the production of the acute acting toxin could only be examined in a few of the 32 cholera-like vibrios. The result was the same as for the haemotoxins. All of them secreted the soluble acute acting toxin and antisera showed cross-neutralisation. Thus, Kraus and his coauthors grant an exceptional position to the El Tor vibrios. In common with V. cholerae they shared morphological and cultural characteristics, as well as agglutination by cholera antiserum and a positive Pfeiffer reaction. They differed by the production of haemolysin and soluble toxin. Later Kraus (1909) had re-examined the El Tor vibrios and declared that the strains haemolytic on sheep blood agar plates were not responsible for epidemic cholera. He called them ‘paracholera vibrios’ and believed that they were able to cause only sporadic cases of a cholera-like disease. The distinction between the haemolytic and haemodigestive activity of the El Tor vibrio is recalled in detail by Pollitzer (1959) who recognises the important contributions of Schumacher and van Loghem using plates with whole blood and lysed blood. El Tor showing clear zones of clearing on the lysed blood, but not V. cholerae. If haemolysis in vibrios caused uncertainty in studies between the V. cholerae biotypes one hundred years ago, then they continue to intrigue workers today. The identity of the El Tor haemolysin has since been shown to be a pore-forming toxin (HlyA) but a further haemolysin present in O1 V. cholerae has been identified (discussed in (Zhang and Austin, 2005)). In failing to identify a soluble toxin in V. cholerae Kraus confirmed earlier work of Richard Pfeiffer (1894, 1895a, b) but the evidence of an additional toxin gave the El Tor vibrios equal if not greater significance than Koch’s comma vibrio. However, many years elapsed before it became clear that Felix Gotschlich’s El Tor vibrio is a biotype of V. cholerae, distinct from the classical biotype with the general scientific perception being that El Tor vibrios were not as virulent as Koch’s V. cholerae. The delay in realisation that El Tor strains were toxigenic and able to cause epidemic cholera meant that International Sanitary Regulations resulting from
the 15th World Health Assembly meeting only recognised El Tor strains as true cholera in 1962, triggered by the start of the 7th cholera pandemic in Indonesia in 1961 (Kamal, 1974). The exhaustive treatise on cholera by Pollitzer in 1959 typified the viewpoint of many that El Tor strains were non-pathogenic as late as 1959. [Intruigingly, Kraus in 1909 had proposed that the El Tor strains were responsible for epidemic cholera (Kraus, 1909).] The early studies on the acute soluble toxin reported by Kraus are difficult to interpret because of contaminating endotoxin. The toxin was considered for many years to be intracellular and only released upon lysis of the bacterial cell (a problem in common with studies of Shigella toxins of the period as well). Nevertheless, it remains sobering that the elucidation of the mode of action of cholera toxin took another 50 years. Whilst the name El Tor is perpetuated alongside cholera Gotschlich, Kraus and Ruffer all considered a role for the organism in dysentery. Dysenteric disease was a far more frequent cause of morbidity and mortality than cholera at the El Tor quarantine camp. Table 2 shows how infrequent cholera outbreaks actually were in Egypt. Having drawn the work of Gotschlich and others to one’s attention, can we confidently explain how Vibrio cholerae El Tor strains were isolated from people without cholera? Our understanding of the sequential series of phage infections, of which one codes for the classic cholera toxin provides a theoretical framework around which we can now understand the isolation of non-toxigenic Vibrio cholerae-like organisms from rivers (Faruque and Mekalanos, 2003). The carrier state had been identified by 1906 and it remains the case that V. cholerae rarely adopts a long lived carrier state.
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Werner Ko¨hler Adolf-Reichwein-Str. 26, D-07745 Jena, Germany Simon P. Hardy Norwegian Veterinary School, PO Box 8146, N-0033 Oslo, Norway