Nobel Lecture, December 11, 1907
Protozoa as Causes of Diseases
My scientific colleagues of the Caroline Institute having done me the very great honour of awarding me the Nobel Prize in Medicine this year for my work on diseases due to Protozoa, the regulations of the Nobel Foundation oblige me to give a summary of my main researches on this question.
I must however go back a little in order to explain how I was led to concern myself with the pathogenic protozoa.
In 1878 after having finished my course of instruction at the School of Military Medicine of Val-de-Grâce, I was sent to Algeria and put in charge of a department of the hospital at Bone. A large number of my patients had malarial fevers and I was naturally led to study these fevers of which I had only seen rare and benign forms in France.
Malaria which is almost unknown in the north of Europe is however of great importance in the south of the Continent particularly in Greece and Italy; these fevers in many of the localities become the dominant disease and the forms become more grave; alongside the intermittent forms, both the continuous forms and those called malignant appear. In the tropical and subtropical regions, endemic malaria takes first place almost everywhere among the causes of morbidity and mortality and it constitutes the principal obstacle to the acclimatization of Europeans in these regions. Algeria has become much less unhealthy than it was at the commencement of the French occupation but one still comes across regions such as the banks of Lake Fezzara, not far from Bone, in which endemic-epidemic malaria rages every year.
I had the opportunity of making necropsies on patients dead from malignant fever and of studying the melanaemia, i.e. the formation of black pigment in the blood of patients affected by malaria. This melanaemia had been described by many observers, but people were still in doubt about the constancy of the alteration in malaria, and about the causes of the production of this pigment.
I was struck by the special characters which these pigment grains presented especially in the capillaries of the liver and the cerebrospinal centres, and I tried to pursue the study of its formation in the blood of persons affected by malarial fever. I found in the blood, leucocytes more or less loaded with pigment, but in addition to these melaniferous leucocytes, pigmented spherical bodies of variable size possessing amoeboid movement, free or adherent to the red cells; non-pigmented corpuscles forming clear spots in the red cells; finally pigmented elements, crescentic in shape attracted my attention, and from then on I supposed they were parasites.
In 1880 at the Military Hospital at Constantine, I discovered on the edges of the pigmented spherical bodies in the blood of a patient suffering from malaria, filiform elements resembling flagellae which were moving very rapidly, displacing the neighbouring red cells. From then on I had no more doubts of the parasitic nature of the elements which I had found; I described the principal appearances of the malarial haematozoon in memoranda sent to the Academy of Medicine, the Academy of Sciences (1880-1882) and in a monograph entitled: Nature parasitaire des accidents de l’impaludisme, description d’un nouveau parasite trouvé dans le sung des malades atteints de fièvre palustre, Paris, 1881.
These first results of my researches were received with much scepticism.
In 1879, Klebs and Tommasi Crudeli had described under the name of Bacillus malariae, a bacillus found in the soil and water in malarial localities and a large number of Italian observers had published papers confirming the work of these authors.
The haematozoon which I gave as the agent of malaria did not resemble bacteria, and was present in strange forms, and in short it was completely outside the circle of the known pathogenic microbes, and many observers not knowing how to classify it found it simpler to doubt its existence.
In 1880, the technique of examination of the blood was unfortunately very imperfect, which contributed to the prolongation of the discussion relative to the new haematozoon and it was necessary to perfect this technique and invent new staining procedures to demonstrate its structure.
Confirmatory investigations at first rare, became more and more numerous; at the same time endoglobular parasites were discovered in different animals which closely resembled the haematozoon of malaria. In 1889, my haematozoon had been found in the majority of malarial regions and it was not possible to doubt any more either its existence or its pathogenic role.
Many observers before me had sought without success to discover the cause of malaria and I should also have failed if I had been content merely to examine the air, water, or the soil in malarial localities as had been done up till then, but I had taken as the basis of my investigations the pathological anatomy and the study in vivo of malarial blood and this is how I was able to reach my goal.
The malarial haematozoon is a protozoon, a very small protozoon since it lives and develops in the red blood cells which in man have a diameter of only 7 microns.
One can summarize the principal ways in which the parasite appears in human blood, as follows:
1. Small, rounded, non-pigmented elements, 1-2 microns in diameter, forming clear spots in the red cells. In stained preparations a nucleus can be detected in each of these small elements. Multiplication is by halving or by multiple division.
2. Amoeboid elements inside the red cells or adherent to their surface, of variable form and dimensions and containing blackish pigment. The largest of these elements are the size of leucocytes. In stained preparations a nucleus can be detected in each amoeboid body. The parasitized red cells change: become paler and increase in diameter, and finally disappear. The gravity and constancy of the anaemia produced by these parasites, which develop rapidly at the expense of the red cells, is thus explained. Multiplication is by multiple segmentation (rose petal or segmented body).
3. Crescentic-shaped bodies measuring 8-9 microns long which are more or less tapered at the ends; towards the middle a corona of grains of pigment can be seen surrounding a nucleus which can only be detected in heavily stained specimens.
4. Flagellae. When a preparation of malarial blood is examined during a feverish attack one can often see developing on the edges of the pigmented spherical bodies derived from the amoeboid or crescent-shaped bodies, very fine flagellae, 20-25 microns long. These flagellae move actively, displacing the neighbouring red cells, and end by becoming detached; and once free they lose themselves among the mass of red cells.
The role of these flagellae was still not decided when the researches of Simond, Schaudinn and Siedlecki showed that analogous elements are found in the Coccidia and that they are male elements destined to fertilize the female elements.
It has now been definitely proved that the haematozoon of malaria has, like the Coccidia, two forms of reproduction: asexual, represented by the segmented bodies, and sexual, the flagellae being the male elements.
A large number of observers have admitted the existence of several species of malarial haematozoa, but for my part I have always defended the unity of malaria and its haematozoon, and the postulated species described under the names of parasites of tropical malaria, aestivo-autumnal fever, tertian or quartan fever appear to me to constitute simple varieties of the same haematozoon.
After the discovery of the malarial parasite in the blood of the patients an important question still remained to be solved: in what state does the haematozoon exist outside the body and how does infection occur? The solution of this problem required long and laborious researches.
After having vainly attempted to detect the parasite in the air, the water, or the soil of malarial areas and trying to cultivate it in the most varied media, I became convinced that the microbe was already present outside the human body in a parasitic state and very probably as a parasite of mosquitoes.
I put forward this opinion in 1884 in my Traité des fièvres palustres (Treatise on paludal fevers) and I returned to it on several occasions.
In 1894, in a report to the International Congress of Hygiene at Budapest on the aetiology of malaria, I wrote: “The failure of attempts at culture have led me to believe that the microbe of malaria lives outside the body in the parasitic state and I suspect in the mosquitoes which are abundant in malarial areas and which already play a very important role in the propagation of filariasis.” This opinion on the role of mosquitoes was considered by most observers at this time as not very likely.
In 1892 two Italian authors who have since become great supporters of infection by mosquitoes wrote:”Laveran supposes that mosquitoes are the intermediate hosts of the malarial parasite. We must object that mosquitoes do not attack birds and in addition, that there are many healthy localities where mosquitoes are abundant. Apart from these objections Calandruccio has observed that the parasites of malaria die in the intestine of the mosquitoes without further development. Laveran’s opinion rests therefore without any foundation, and the hypothesis put forward by us that the parasites exist outside the body in the form of amides is confirmed.”1
King2 in America had suggested in 1883 that mosquitoes played a part in the aetiology of malaria but he did not know of my work on the haematozoon of malaria and could not specify what part the mosquitoes played, so that in saying that the mosquito acted as a temporary host of the malarial parasite I had obviously got closer to the problem than King had been able to do. I indicated clearly the route that it was necessary to follow to arrive at the goal; to seek out what became of the parasite in the body of mosquitoes which had sucked malarial blood.
Having left the malarial countries it was not possible for me to verify the hypothesis I had put forward on the role of mosquitoes, and it is to Dr. Ronald Ross that we owe the demonstration that the malarial haematozoon and the closely related Haemamoeba malariae of birds complete several phases of their evolution in the Culicidae and are propagated by these insects.
R. Ross whose excellent and patient investigations have been very justly rewarded by being given the Nobel Prize in Medicine in 1902, has been very willing to acknowledge in many of his papers that he had been usefully guided by my deductions and by those of P. Manson.
Today the transformations which the malarial parasite undergoes in mosquitoes of the genus Anopheles are well known and no further doubt is possible on the role these insects play in the propagation of malaria. I consider it is useless to insist on this as it has been very completely dealt with by R. Ross in a conference held here in 1902.
From 1899, my principal work on malaria has had as the object, the study of the Culicidae in their relation to malarial endemicity and of the rational prophylaxis of this dreadful malady.
I have studied the Culicidae of France and of the French colonies and I have been able to show in a series of annotations published at the Academy of Sciences, or at the Academy of Medicine, and at the Society of Biology that Anopheles can be found in all the malarial localities.
Dr. Battesti and I have demonstrated that Anopheles could be found in all the malarial localities of Corsica which had been considered free from these Culicidae.
On my suggestion leagues against malaria have been set up in Corsica and in Algeria. These leagues have already rendered great service, the principal aim which they have to strive for being to spread among the public the new ideas concerning the cause, the mode of transmission, and the rational prophylaxis of malaria.
This year I have published the second edition of my Traité du paludisme and in the Introduction I have been able to write: “Malaria, the history of which not long ago presented such obscurities, is today one of the best known diseases. The study of the clinical forms and the pathological changes has been completed and defined; the haematozoon which I described in 1880 has been recognized by all writers as the cause of malaria; its propagation by mosquitoes has been demonstrated, and now we are able to fight the malady by specific drug treatment and rational prophylaxis.”
I come now to the examination of my other work on the intracellular haematozoa, the number of which has increased continually since the discovery of Haemamoeba malariae.
As from 1889, I have classified the endoglobular haematozoa or Haemocytozoa in three genera: Haemamoeba, Piroplasma, Haemogregarina.
In the genus Haemamoeba, I have studied the haemamoebae of birds which closely resemble H. malariae and I have described several new species in Padda oryzivora, in a titmouse, in the partridge and the turkey.
In 1889, I studied with Nicolle P. bigeminum and P. ovis in the genus Piroplasma and described the forms of multiplication of these haematozoa.
In 1901, I described Piroplasma equi and in 1903, the bacilliform piroplasma of the Bovidae from preparations which had been sent to me by Mr. Theiler, a Transvaal veterinary surgeon.
In 1903 and 1904, Mesnil and I helped to make known, under the name of Piroplasma Donovani, the parasite of Kala-Azar discovered in India by Doctors Leishman and Donovan.
In the genus Haemogregarina, I studied the endoglobular parasites of Chelonians, Bactrachians, Saurians, Ophidia, and fishes.
I described the forms of endogenous multiplication of the haemogregarines of Chelonians and reported a series of new species.
From 1897 to 1900, I published either by myself or with the collaboration of Mesnil, a series of notes on the Sporozoa, properly so-called: Coccidia and Myxosporidia, of which I reported several new species, Sarcosporidia and Gregarines.
In recent years I have devoted myself to the study of the trypanosomes which are the cause of a large number of epizootics: Surra, Nagana, Dourine, Souma, etc.; the pathological importance of these parasites has increased especially since it has been shown that the grave endemic disease known in Equatorial Africa under the name of sleeping sickness, was produced by a trypanosome Tr. gambiense.
Trypanosomes are Protozoa, very different from the endoglobular haematozoa; they live in the free state in the plasma and not in a state of inclusion in the red cells or in other anatomical elements; they belong to the class of Flagellidae.
The body protoplasm of a trypanosome is generally fusiform and more or less tapered at the ends. When stained, two masses of chromatin can be seen: a large one towards the middle of the body, the nucleus; the other, small, situated usually near the posterior end, the centrosome. The flagella which starts from the centrosome and bounds the undulating membrane usually ends at the anterior extremity by a free portion which forms the flagellaproper.
I have published a large number of notes and memoranda on the trypanosomes, and in 1904, with Mesnil a monograph entitled: Trypanosomes et Trypanosomiases.
Among the original investigations carried out either by myself or in collaboration, I should like to mention: investigations on the structure of trypanosomes and of that of the Flagellidae in general; the agglutination of trypanosomes and the conditions which produce it; the differentiation of the trypanosomiases; the trypanosomes of rats, Tr. Lewisi, of Nagana, Tr. Brucei, of horses in the Gambia, Tr. dimorphon; of sleeping sickness Tr. gambianse, one affecting bovines in the Transvaal Tr. Theileri, on three new species pathogenic for equines and bovines of the Upper Niger, Tr. Cazalboui, Tr. Pecaudi, and Tr. soudanense; on new trypanosomes of birds, Chelonians, Bactracians or fishes.
As far as fishes are concerned Mesnil and I have established the existence not only of new species but of a new genus, Trypanoplasma.
Trypanoplasma have been found in the rudd, carp, and minnow. Their structure differs markedly from that of the trypanosomes as can be ascertained from well-stained specimens. The body protoplasm is generally crescentic; there are two chromatin masses, the larger one, the nucleus, on the side of the convexity, the other narrower, more deeply stained situated usually on the edge of the concavity, the centrosome. This gives rise to two flagellae, an anterior one which becomes free at once, and one which borders the undulating membrane and only becomes free at the posterior extremity.
I have published several notes on the distribution of the Glossinae (tsetse) in Equatorial Africa and of other biting flies capable of propagating the trypanosomiases.
Finally, I should mention a series of papers on the prophylaxis and treatment of diseases due to trypanosomes. In 1904, I showed that arsenious acid (in the form of sodium arsenite) produced excellent effects in infections due to Tr. gambiense; and in 1904 and 1905, I communicated several notes to the Academy of Sciences on the combined treatment of various trypanosomiases by arsenious acid and Trypan red. With this method of treatment I have had good results in mice, rats, dogs, and monkeys infected with Tr. Evansi, Tr. gambiense, Tr. equiperdum; unfortunately later investigations have shown that Trypan red is badly tolerated by man.
With Dr. Thiroux. I undertook new experiments aimed at investigating whether the combination of two different arsenical preparations would not give better results than the use of arsenious acid alone or atoxyl alone. The results which we obtained by combining atoxyl with arsenic trisulphide or arsenious iodide have been very satisfactory and we hope that this new drug treatment may be used in human trypanosomiasis or sleeping sickness which is raging at the present time among the natives of Equatorial Africa and of which there are more and more cases among Europeans.
To summarize: for twenty-seven years, I have not ceased to busy myself with the study of the parasitic Protozoa of man and animals and I can say, I believe without exaggeration, that I have taken an important part in the progress which has been made in this field.
Before the discovery of the malarial haemotozoon no pathogenic endoglobular haematozoon was known; today the Haemocytozoa constitute a family, important for the number of genera and species and also for the role some of these Protozoa play in human or veterinary pathology.
By directing the attention of doctors and veterinary surgeons to examination of the blood, study of the endoglobular haematozoa prepared the way for the discovery of the diseases due to trypanosomes which themselves also constitute a new and very important chapter in pathology.
The knowledge of these new pathogenic agents has thrown a strong light on a large number of formerly obscure questions. The progress attained shows once more how just is the celebrated axiom formulated by Bacon: “Bene est scire, per causas scire.”
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