Development Of Protozoology

The development of this discipline was totally dependent on the introduction of satisfactory microscopes (Cole, 1926). Although Gesner was probably the first to visualise a protozoan parasite in 1565, it was a century later that Robert Hooke (1635-1703) produced a diagram in his Micrographia. The birth of protozoology as a science was, however, due to van Leeuwenhoek (Dobell, 1932) (Figure 1.4) who, in 1674, visualised free-living ciliates in fresh water; he later described cysts of Eimeria stediae in rabbit bile. In 1680, the same worker observed motile 'animalcules' in the gut of a horse-fly, and in 1681 in his own stool; these were almost certainly Giardia lamblia.

Antony van Leeuwenhoek was born in the small Dutch town of Delft. Lacking scientific training, he became a respected local tradesman (he ran a small haberdashery business) but had sufficient leisure time to devote to scientific pursuits. He made his own lenses and microscopes, through which he originally observed 'animalcules' in marshy water. Most of his results were communicated to the Royal Society in London, to which he was duly elected. van Leeuwenhoek wrote a great deal, and his last letter was written in 1723, his 91st year. He was without doubt the 'father of protozoology'.

More than 100 years were to pass before further parasitic protozoa were recorded, although many free-living forms were described during this time. The term 'Protozoa' was probably introduced about 1820; shortly after this C. G. Ehrenberg (1795-1876) and Felix Dujardin (1801-1860) published important texts on the subject. Various protozoa of insects and fish received a great deal of attention at this time. In 1836, Alfred Donne (1801-1878) discovered Trichomonas vaginalis and in 1858 a probable case of coccidiosis, accompanied by a postmortem report, was published. Around the mid-nineteenth century, a number of human intestinal flagellates were documented, and in 1856 Pehr Malmsten (1811-1883) of Stockholm, described what was probably Balantidium coli. The first major pathogenic protozoan of Homo sapiens to be described was Entamoeba histolytica, which was described by Losch (see below) in 1873.

Entamoeba histolytica

James Annesley (1780-1847) of the East India Company, was aware of two forms of dysentery. In his classic two-volume work, Researches into the Causes, Nature and Treatment of the More Prevalent Diseases of India ...(1828) he clearly differentiated between what were to become known as amoebic colitis and shigellosis; he associated the former with hepatic problems (including 'abscess of the liver'). Losch recorded his observations in Virchow's Archiv for 1875, but did not recognise that some E. histolytica were pathogenic whereas others were not (as later suggested by Emile Brumpt [1877-1951]), and furthermore he considered that this organism was not the cause of dysentery but acted as an 'irritant', thus preventing the colonic ulcers (caused by another agent) from healing. Following this observation, Robert Koch (1843-1910), who was carrying out his researches in Egypt into cholera in 1883, noted E. histolytica in both the colon and liver abscess; he was meanwhile too interested in cholera to pursue this organism, but his observation acted as a catalyst for Staphanos Kartulis (1852-1920), who was working in Alexandria, and in 1887 demonstrated the organism in necrotic tissue of a liver 'abscess'; in 1904, he published an account of E. histolytica in a cerebral 'abscess'. The results of Kartulis's studies were published in Virchow's Archiv and attracted the attention of William Osler (1849-1919), at that time working in Baltimore (Cook, 1995). Heinrich Quincke (1842-1922) and Ernst Roos (1866-?) meanwhile described the cystic form of

Protozoology Babes

Fig. 1.4 Antony van Leeuwenhoek (1632-1723), the founder of protozoology, who probably visualised Giardia lamblia in his own faecal sample. Reproduced by courtesy of the Wellcome Institute Library, London this protozoan parasite, which they showed was infective to cats when given by mouth. At the commencement of the twentieth century, the role of E. histolytica in dysentery was far from clear; however, in 1903 Leonard Rogers (1868-1962) published a paper from Calcutta, in which he described how the organism(s) spread from gut to liver via the portal veins. As late as 1909, however, Manson was not totally convinced that E. histolytica was the cause of 'tropical dysentery'.

Ernest Walker (1870-1952) working in Manila, The Philippines, between 1910 and 1913 again suggested that there were two forms of E. histolytica, one pathogenic and the other not. During the First World War (1914-1918), C. M. Wenyon (1878-1948), working in Alexandria, emphasised the importance of the 'carrier state'. Clifford Dobell (1886-1949) published his classic monograph, The Amoebae Living in Man, in 1919.

Babesia spp.

Elucidation of the life-cycle of Babesia spp. the cause of Texas Fever (in cattle) is of interest (Foster, 1965), although this organism is not of great practical importance. Theobold Smith (1859-1934) a pupil of Daniel Salmon (18501914) (of Salmonella fame) together with Frederick Kilborne (1858-1936), published Investigations into the Nature, Causation and Prevention of Texas or Southern Cattle Fever (1893). The disease seemed to be caused by an intra-erythrocytic protozoan parasite, a finding that did not fit into any of the then known classifications. Furthermore, transmission seemed to be associated with a tick (Ixodes bovis); details of the development of the parasite (in the tick) were not finally worked out until some 40 years after Smith's work. In 1888, V. Babes (who in fact gave his name to babesiosis) had previously visualised an intra-erythrocytic protozoan in affected cattle in Romania.

Plasmodium spp. and 'the Great Malaria Problem' (Cook, 1997b)

In the latter years of the nineteenth century, the cause of malaria (and its treatment) had not progressed since the introduction of cinchona bark, a specific for the 'intermittent fevers'. The fact that malaria is transmitted by the bite of mosquitoes had been suspected for many centuries (Cook and Webb, 2000). Mosquito nets were in fact used in ancient Rome to prevent 'the fever'. Furthermore, there are suggestions in writings over several centuries that the mosquito was indeed involved; for example, in 1717 Giovanni Lancisi (1654-1720), physician to the Pope and a professor at the Sapienzia in Rome, suggested this form of transmission, whilst at the same time accepting the miasmatic theory for transmission of disease. In 1716, Lancisi had demonstrated 'grey-black pigment' in malaria tissue. In 1882, Dr Albert Freeman Africanus King (1841-1914) read a paper to the Philosophical Society of Washington, suggesting (on epidemiological grounds) that Plasmodium was transmitted by the bite of the mosquito. It was not until 1880 that Alphonse Laveran (18451922), recipient of the Nobel prize for 'medicine or physiology' in 1907 working in Algeria, demonstrated Plasmodium in the human erythro-cyte (Bruce-Chwatt, 1988; Cook, 1993a); on 6 November of that year he visualised several long flagella being extruded from a hyaline body in a 24 year-old artilleryman. In 1885, Camillo Golgi (1843-1926) was able to show that in malaria, 'fevers' correlated with the liberation of mero-zoites into peripheral blood; he showed furthermore, that tertian and quartan fevers were caused by different parasites. Ettore March-iafava (1847-1935) and Amico Bignami (18621929) were the first to distinguish P. falciparum from the 'benign' malarias. In 1893, Bignami and Giuseppe Bastianelli (1862-1959) showed, by inoculating volunteers with blood known to contain Plasmodium spp., that 'fever' was always caused by the 'young' parasite, and never the 'crescent' (the sexual form, or gametocyte). By 1890 it was widely accepted that Laveran's parasites were the cause of malaria (Cook, 1995).

In three classical Goulstonian Lectures delivered to the Royal College of Physicians of London in 1896, Manson (in the light of his filaria researches; see above) spelled out his mosquito-malaria hypothesis (which he had first formulated in 1894) in great depth (Cook, 1993a). This, without doubt, formed the stimulus

Protozoology Babes
Fig. 1.5 Ronald Ross (1857-1932) who established the role of the mosquito in transmission of Plasmodium spp. and elucidated the complete life-cycle of avian malaria (Proteosoma spp.) in Secunderabad and Calcutta, India, respectively

for the subsequent researches of Ronald Ross (1857-1932) (Bynum and Overy, 1998).

Ross (Figure 1.5) had been born in India. His father, of Scottish descent, was a general in the Indian Army. Ross first became interested in malaria in 1889. After discussions with Manson, who subsequently became his mentor (Bynum and Overy, 1998), he worked on human malaria in India; however, he failed to produce infection in volunteers by the bites of Culex or Aedes mosquitoes, but demonstrated malaria pigment in a mosquito at Secunderbad on 20 August 1897 ('mosquito day'). He was then posted to a region where he was not able to study human disease, and therefore turned his attention to avian malaria (Proteosoma spp., which is transmitted by the bite of Culex). By a series of careful experiments begun in 1897, he demonstrated the bird-mosquito-bird cycle of this protozoan parasite in 1898; the culmination of this work came on 4 July of that year (Bynum and Overy, 1998). These observations were communicated by Manson to the British Medical Association's meeting, held in Edinburgh on 28 July 1898. Also in 1898 (November-December), Amico Bignami, Guiseppe Bastianelli and Battista Grassi (see above) were able to demonstrate the man-mosquito-man cycle in a series of experiments carried out in Italy; this work was confirmed by Ross in Sierra Leone in 1899. However, because malaria was endemic in both Italy and Sierra Leone, neither study could possibly be definitive, because a new infection might easily have been introduced. In 1900, Manson initiated two experiments in order to clinch the man-mosquito-man component of the cycle. A team consisting of Low (see above), Louis Sambon (1865-1931), Signor Terzi (an artist) and a servant slept in a mosquito-proof hut in the Roman Campagna, approximately 8 km from Rome, for a period of 3 months (19 July-19 October 1900); they lived normal outside lives during the course of the day, but did not become infected with malaria. In the second experiment, it was arranged (with the collaboration of Bastianelli) to send mosquitoes infected with P. vivax from Rome to London in a mosquito box (as late as the 1920s and 1930s P. vivax infection was common in the Roman Campagna). On arrival in London, the surviving specimens were allowed to feed on P. T. Manson (1878-1902) (Manson's elder son) and a laboratory technician (George Warren). In both cases, clinical malaria developed; the former subsequently experienced two relapses following quinine chemotherapy. The two experiments were published, like so many early major discoveries in clinical parasitology, in the British Medical Journal—for 1900.

Despite his successes, Ross was an extremely difficult individual with whom to work; evidence has been summarised by Eli Chernin (Cook, 1993a). For example, Manson was requested to write a testimonial for a Dr Prout who had applied for Ross's post in Liverpool, which had become vacant in 1912 after his removal to London. He made two comments to which Ross took great exception: 'I sincerely hope that his appointment may be successful, for it would, if I

may use the expression, make good a defect in your system of teaching ...' and, furthermore, 'A teacher of Tropical Medicine, to be considered efficient, should be not only a scientific man, but one having had extensive experience in tropical practice'. Manson was, either consciously or subconsciously, highlighting the fact that Ross was not a great clinician, even though his scientific work was satisfactory. As a result, Ross sought legal advice, the matter being narrowly resolved without a court case. It seems exceedingly ungrateful of Ross to have pursued this libel action against his mentor who was, in effect, largely responsible for an FRS and Nobel Prize (Cook, 1993a); however, this merely reflects the eccentric nature of Ross, who has variously been described as '. . . capable of magnifying a petty affair out of all proportions', 'chronically maladjusted', or 'a tortured man' (Cook, 1993a).

It was not until the early 1940s that Neil Hamilton Fairley (1891-1966) clearly demonstrated the non-haematogenous phase in the life cycle of Plasmodium spp. (Cook, 1993a). He observed that a parasitaemia was present in peripheral blood immediately after infection, but that this disappeared during the incubation period of the disease. In 1948, Henry Shortt (1887-1987) and Percy Garnham (1901-1993) were able to demonstrate the 'hypnozoite' phase of P. vivax within the hepatocyte, thus putting a seal on the life-cycles of all human (and monkey) Plasmodium spp. infections recognised at that time.

The first attempt(s) at malaria prophylaxis by prevention of anopheles mosquito bites was made by Angelo Celli (1857-1914) in 1899.

Therefore, by the end of 1900, the life-cycles of two vector-borne parasitoses, one helminthic and the other protozoan—lymphatic filariasis and Plasmodium spp. infection—had been clearly delineated (see above, Cook, 1993a). In the same year, mosquito transmission of the viral infection yellow fever (see above), was also clearly demonstrated, this time by American workers. The major figures in this breakthrough were Carlos Finlay (1833-1915) and Walter Reed (1851-1902) (Cook, 1993a). However, it seems most unlikely that this discovery could have taken place in the absence of the foregoing British work.

Trypanosomiasis: Slow Elucidation of the Cause

African Trypanosomiasis

David Livingstone (1813-1873) had been convinced in the mid-nineteenth century that the tsetse fly was responsible for transmission of 'nagana', a disease which affected cattle in Central Africa. This is clearly recorded in his classic Missionary Travels, first published in 1857; there is, in this work, an accurate drawing of the tsetse fly. It seems probable that he had in fact associated the bite of Glossina palpalis with 'nagana' as early as 1847. It was not until 1894, however, that the causative role of Trypanosoma (later designated T. brucei) was delineated in nagana and this resulted from David Bruce's (Figure 1.6) brilliant work in Zululand, where he had been posted from military duty in Natal (Cook, 1994). Shortly before this, animal trypa-nosomes had been visualised, and in 1878 Timothy Lewis (see above) had first indicated that trypanosomes could cause infection in mammals.

A febrile illness associated with cervical lymphadenopathy and lethargy had been clearly recorded in Sierra Leone by T. M. Winterbottom (1765-1859) in 1803. In 1902, Joseph Dutton

Fig. 1.6 David Bruce (1855-1931), who established the causes of nagana (in Zululand) and the 'negro lethargy' (in Uganda)

(1874-1905) (Braybrooke and Cook, 1997) and John Todd (1876-1949) demonstrated that Trypanosoma spp. were responsible for this condition, then named 'trypanosome fever' in West Africa; their observations were made on an Englishman who had been infected in the Gambia. Studies were carried out in both the Gambia and Liverpool. This work was published in 1902 with a full clinical description, accompanied by temperature charts.

Early in the twentieth century an outbreak that was described at the time as 'negro lethargy' swept Central Africa; this involved the northern shores of Lake Victoria Nyanza (Cook, 1993b). No-one, it seems equated the disease with 'trypanosome fever'. In 1902, the Royal Society sent a Sleeping Sickness Expedition, consisting of Low (see above), Aldo Castellani (1877-1971) and Cuthbert Christy (1864-1932) in an attempt to determine the aetiological agent responsible for this disease. Manson was of the opinion that Filaria perstans was responsible; he had visualised this parasite in three cases of sleeping sickness investigated in London, at the London and Charing Cross Hospitals. After a great deal of painstaking work, Castellani concluded that the disease was caused by a streptococcus. He reported his finding to the Royal Society's Malaria Committee, chaired by Joseph Lister (1827-1912), but they were far from enthusiastic. In the meantime, Castellani had visualised Trypanosoma spp. in the cerebrospinal fluid of a single case of 'negro lethargy'; however, he disregarded this organism, and favoured the streptococcal theory. The Royal Society proceeded to send a second team to Uganda in 1903, consisting of Bruce (Figure 1.6) (Cook, 1994) and David Nabarro (1874-1958). They demonstrated Trypanosoma spp. in numerous cases of sleeping sickness (in both cerebrospinal fluid and blood) and furthermore, were able to transmit T. gambiense to monkeys via the bite of infected Glossina palpalis (the local species of tsetse fly); this work clinched the aetiological agent responsible for this disease.

Castellani remained convinced, however, that he should be given credit for discovering the cause of sleeping sickness, now correctly attributed to Bruce and Nabarro. Acrimonious correspondence emerged, some being recorded in The Times for 1908 (Cook, 1993b). In retro spect, it seems likely that Castellani was unduly influenced by a report from some Portuguese workers which concluded that a diplo-streptococcus was responsible for the disease; Castellani, a trained bacteriologist, was clearly far more impressed with this organism than with Trypanosoma spp.!

Several years were to pass before the animal reservoirs of African trypanosomiasis were delineated. Was the causative organism of nagana identical with that which caused African trypanosomiasis? It was not until 1910 that J. W. W. Stephens (1865-1946) and H. B. Fantham (1875-1937) discovered T. rhodesiense in Nyasaland (now Malawi) and Northern Rhodesia (now Zambia). In 1911, Allan Kinghorn (?-1955) and Warrington Yorke (1883-1943) demonstrated the transmission of T. rhodesiense to man by Glossina morsitans.

South American Trypanosomiasis

Human South American trypanosomiasis was first recorded in 1910. Carlos Chagas (18791934), working in a remote part of Brazil, became aware that a high proportion of houses were infected with the reduviid bug (the 'kissing bug'), which bit at night. The bug harboured an organism (which developed in the gut and migrated to the proboscis for subsequent inoculation) which was infective to monkeys and guinea-pigs. Chagas showed, furthermore, that an acute febrile illness in children (characterised by oedema, especially of the eyelids, anaemia and lymphadenopathy) was caused by this organism. In 1917 Torres described the cardiac lesions of Chagas' disease. Recognition of the 'mega' syndromes followed. That faecal material from the bug caused infection had been suggested by Chagas, but demonstrated conclusively by Dias (Foster, 1965) in the early 1930s.

Visceral Leishmaniasis (Kala Azar): a Disease with a Potential Influence on the 'Jewel in the Crown'—India

The protozoan parasite responsible for kala azar (or 'dum-dum' fever) has a patchy distribution throughout tropical and sub-tropical countries (Cook, 1993a). The causative agent was initially demonstrated by William Leishman (1865-1926) at the Royal Victoria Hospital, Netley (off Southampton Water) in 1900. He did not publish this work until May 1903. In April of that year, Charles Donovan (1863-1951), working at Madras, India, confirmed the observation. The parasite was subsequently named the Leishman-Donovan body (now designated the amastigote of Leishmania donovani).

A related agent, L. tropica, the causative agent of cutaneous leishmaniasis (Delhi boil), was first demonstrated by J. H. Wright (1870-1928), also in 1903; this organism had incidentally been described 5 years earlier by a Russian worker, P. F. Borovsky (1863-1932). Rogers (see above), a great physician who, amongst many contributions to clinical parasitology and tropical medicine, founded the Calcutta School of Tropical Medicine in 1920, first cultivated 'Leishman-Donovan bodies' in Calcutta in 1904. It was not until 1911, however, that Wenyon (see above) was able to demonstrate transmission of (cutaneous) leishmaniasis by Phlebotomus spp. (the sandfly).

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