Researches on malaria Nobel Lecture, December, 12, 1902
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of Laveran’s discovery, which was then slowly fighting its way into recogni- tion. A fuller account of his excellent paper is given in Nuttall’s history 65,66 .
R E S E A R C H E S O N M A L A R I A 33 Laveran’s conjecture was first given briefly in 1884 3 , evidently independ- ently of King. Seven years later he mooted the same idea, still very briefly and without giving many reasons 11 . The similar conjecture of Koch was not pub- lished at all; but in a letter to me he says that the mosquito theory occurred to him during his first visit to India in the winter of 1883-1884, and that R. Pfeiffer mentioned the matter publicly in 1892 (see Koch’s letter in sec- tion 23). As already stated Manson did not arrive at his hypothesis until near the end of 1894, when he drew attention to it (after mentioning it to me) in a short article 22
but upon a very powerful parasitological argument of his own which was as follows. The work of Laveran, Golgi, Marchiafava, Celli and others had clear- Fig. 1. Gametocytes producing motile filaments; the tertian parasite (1-3); the quartian parasite (4-6); the aestivo-autumnal parasite (7-12). From Manson’s paper, p. 644. 26 34 1 9 0 2 R . R O SS ly established that the general life-cycle of the parasites within the vertebrate host consists of a process of schizogony or asexual spore formation, by means of which the organisms proliferate indefinitely in the blood. But in addition to the sporocytes existing for this purpose, all observers from the time of La- veran had observed certain large cells, which, while they were evidently ma- ture as regards size, did not produce spores and appeared to have no function within the body. Laveran showed, however, that a few minutes after blood containing these cells was withdrawn from the circulation they underwent a singular change - that is, they gave issue to a number of long, actively motile filaments, capable of separating themselves entirely from the parent cell, and progressing independently among the blood corpuscles. There had already been a long discussion about these forms. Grassi, followed by Bignami and many other Italians, considered them to be forms of degeneration, and held that the motile filaments were products of a kind of death agony in vitro. The reason given for this view was that the motile filaments contain no chromatin (which is not true); but in my opinion these observers had not considered them with sufficient attention, or they could not have thought them to be dying bodies. On the other hand Laveran, Danilewsky and Mannaberg, who had studied them closely, came to the opposite conclusion that they constitute in some way the highest stage of the parasite; and Mannaberg even conjected that they may be concerned in the passage from the intracorporeal to the ex- tracorporeal stage of the organisms - though he did not indicate the route by which he thought the passage was made. Manson’s speculation broke in at this point. He accepted Mannaberg’s position; and noted also the general law that parasites must attain some means of passing (at least by their progeny) from the already-infected individual into a fresh individual; that the parasites of malaria being contained within the closed cavity of the circulation cannot escape from it except by the intervention of some external agency (e.g. a suctorial insect); that the position as regards these parasites was indeed the same as that of the filaria embryos which he had shown require the interven- tion of a mosquito to escape from the infected host; and that the epidemio- logical laws of malarial fever suggest a possible connection with the same in- sect. Hence it flashed upon him that the motile filaments mentioned above are really flagellate spores, which, when the parent cells are ingested by the mos- quito, escape and enter the insect’s tissues, developing in them into some form analogous to that of the organisms in the human blood. Manson continued the speculation to a further point, especially in a later publication 26 . It will be remembered by those who have studied his works R E S E A R C H E S O N M A L A R I A 35 that in his original investigations on the development of filaria embryos in mosquitoes he had failed to ascertain that the insects live for more than a few days; he had thought that after a single meal of blood the mosquito lays her eggs on the surface of water and dies in the act of doing so. Consequently, when he had traced the development of the embryos to the stage which they reach in the insect’s thorax he inferred that that was the whole development, and that after the death of the host on the surface of the water the embryos escaped into the latter and finally infected man by the digestive tract.* And he now applied the analogy to the malaria parasites, and thought that, similarly, after the insect’s death, they enter the water and infect man either by drinking water, as he assumed for the filariae, or by the old machinery of the aerial miasma. Thus Manson’s hypothesis suggested a clue only to the departure of the parasite from the human host; it did not attempt to define the route of entry, the exact mode of infection. In these points he admitted that his speculation was looser, and research has shown that he was wrong - certainly with regard to the malarial parasite and probably even with regard to filariae. In another point also he was wrong - the motile filaments are not flagellate spores, as he thought they were. I remember mentioning to him at the time that they might possibly be of the nature of sperms - an idea which was sug- gested to me by Lewis, who conjectured that his trypanosoma might be of that nature 6 . We shall see later what they really are. But these errors were immaterial. The fundamental part of his hypothesis was the close and power- ful argument to the effect that the motile filaments and the parent cells from which they spring must be meant to infect the mosquito in some manner. This was more than a hypothesis; it was a great and illuminating induction. It gave the required clue to further research; and without it I am convinced the ma- laria problem would not have been solved at all and we should still be engaged in a laborious and hopeless search for the parasites in water and air. Cogent as Manson’s arguments appeared to me, they were far from con- vincing to most other students of malaria; and in fact no one else took the trouble to investigate the matter in spite of its immense importance to hu- manity. In 1896 indeed, Bignami wrote a long and dexterous article attacking the whole induction 29 . He still refused to believe that the motile filaments were anything but the result of death in vitro, and added that if the induction were true, malarial fever could be propagated by patients living in the pres- ence of mosquitoes - which he refused to consider possible. At the same time he evolved a theory of his own to the effect that mosquitoes become infected * Or possibly "by piercing the integuments".
36 1 9 0 2 R .ROS S with the parasites when in the larval stage in water, and then inoculate them into man during puncture. Thus while Manson thought mosquitoes carry the parasite from man to the marsh, Bignami thought that they carry it from the marsh to the man. The latter view does not appear to me a very philosophical one, since it presupposes the possibility of organisms living normally partly a saprophytic and partly a parasitic existence in the mosquito, and then being suddenly transferable on exceptional occasions to man. Bignami’s theory, however, was exactly that of King given much more forcibly thirteen years earlier. He mentioned that he had previously attempted to infect men by gnats brought from malarious localities, but that the attempts had failed. He also referred to experiments made by Calandruccio, who had failed to observe any development of the parasites in the stomach of mosquitoes fed on malarial blood. Lastly he cites another most valuable analogy in favour of the mos- quito theory of malaria, namely that of the Pyrosoma bigeminum, a parasite of cattle allied to the parasites of malaria, and known by the brilliant researches of Theobald Smith and Kilborne to be carried by ticks 19 . Koch also had used this analogy, but I think that both Manson and myself had overlooked it some- what unduly.* Curiously enough all this time it seems to have occurred to no one that the mosquito may act in both roles imagined by King and Manson severally - that it may both take the parasite from the patient and also inocu- late it into healthy persons. I traversed Bignami’s criticisms in an article which will be referred to later (section 11). In considering the merits of these various hypotheses we must always re- member that all of them have found no little support from Manson’s original discovery of the development of Filaria bancrofti in mosquitoes.
We must now return to my own labours. As already mentioned, directly I became acquainted with Manson’s induc- tion, I determined to continue my investigation of the malaria problem en- tirely on this new basis. Before my departure for India I discussed with Manson the best method of * The development of the Pyrosoma in ticks is still unknown, though the second host has long been recognized. It should be understood that the history of this organism, and of the filaria in mosquitoes, while adding great force to the mosquito theory of malaria, gave us no information regarding the form and position of the malaria parasites in mosquitoes, nor of the species of insect concerned. The Pyrosoma is not very nearly related to the ma- laria parasites, and the filaria not at all. I was not aware of the work of Smith and Kilborne until much later.
R E S E A R C H E S O N M A L A R I A 37 procedure.* We agreed that the proper course would be to select patients whose blood was rich in gametocytes (the name now given to those forms of the parasite of which some produce motile filaments); to allow mosquitoes to bite these patients; and to attempt to trace in the tissues of these insects the development of the said motile filaments (which we thought were flagellate spores). In fact it was proposed that I should adopt exactly the procedure em- ployed by Manson in regard to Filaria bancrofti. It seemed necessary only to follow the motile filaments, after their escape from the gametocytes contained in the ingested blood in the mosquito’s stomach, to their supposed destination within some kind of cells of the insect’s tissues (e.g. the stomach or blood cells) - apparently an easy task. It was true we anticipated, on the analogy of the filaria, that not all species of mosquitoes would be amenable to the malarial infection, and we recognized that this doubt would increase the difficulties; but we hoped readily to distinguish the proper species by its particular preva- lence in very malarious localities. The motile filaments being traced to their habitat in particular cells of the insect, we thought that it would be easy to observe their further development, and to watch their escape into water after the host’s death. This done we should be able to identify the extra corporeal form of the parasites in water, air, or dew, and to ascertain exactly the route of infection of man.
. I reached India in 1895 and found myself appointed medical officer of a regiment of native soldiers sta- tioned at Secunderabad and suffering much from malarial fever. A survey was immediately made of the malarial parasites existing among these men and I found myself able to confirm for India, in almost every detail, the specialized work of the Italians and of Mannaberg.** * We thought that Manson himself could not undertake the work in England, but this perhaps would not have been as difficult as we supposed. The parasites have now been cultivated in the local Anopheles at Hamburg and have been found in them in Holland. Work might easily have been done in this direction at home, while I was labouring in In- dia. At all events I should have been greatly assisted by a study of British gnats. ** My regiment was stationed near a small marsh and suffered badly, while another regi- ment situated only a mile to leeward of the same marsh escaped. My regiment suffered from the aestivo-autumnal and tertian varieties of parasite, quartan being quite absent; but in the neighbouring regiments of this great garrison quartan abounded - a fact which confirmed me in favour of the view that these varieties are distinct and not interchange- able forms. The work of Crombie and myselfwas the first done in India on this basis, that of Vandyke Carter (1887) being done on the basis of Laveran’s works.
38 1 9 0 2 R . R O S S At the same time I examined the mosquitoes which abounded in the bar- racks and hospital. Before leaving England I had made many attempts to ob- tain literature on mosquitoes, especially the Indian ones, but without success except for some brief notes in encyclopedias; and I did not even clearly recognize the identity of mosquitoes and gnats, but thought that the for- mer constituted a special division of the Culicidae.* Consequently I was forced to rely entirely on my own observations; and I noted that the various species of mosquitoes of the locality belonged apparently to two different groups, separated by many traits, and called these groups for my own con- venience, brindled mosquitoes and grey mosquitoes. It was not until 1897 that I clearly recognized a third group which I called spotted-winged mosquitoes (see sections 12 and 13). As the grey and brindled mosquitoes abounded round the infected barracks, it was naturally thought likely that they were concerned in the propagation of the disease. After some initial difficulties I caused numbers of them (espe- cially the brindled mosquitoes) to be fed on persons with the gametocytes of aestivo-autumnal fever (crescents) in their blood. It should be noted that from the first I employed for this purpose only mosquitoes bred in captivity from the larvae, and not insects caught at random in the houses. There were two excellent reasons for this; first, that the insects caught at random might have already fed themselves previously and have thus, for all I knew, acquired va- rious parasites which might confuse my results; and, secondly, because it is easier to obtain the insects in numbers by collecting their larvae and keeping them in vessels until they hatch out from the pupae, rather than by catching each separately by hand. The mosquitoes were fed by being released from the breeding-jar into a mosquito-net within which the patient was placed, the gorged insects being subsequently caught in bottles and dissected as required. From the first I kept careful notes of my observations, and also recorded them in letters to Manson sent by almost every weekly mail, except when later, being very busy at Bangalore, I was obliged to reduce both notes and letters. The note-books and letters are still in my hands. 9. Secunderabad (1895). The motile filaments in mosquitoes. The first point re- quiring study was the process by which the motile filaments escape from the parent cells (gametocytes) within the stomach cavity of the mosquitoes. The process had been frequently watched in vitro (in slides of liquid blood pre- * In spite of repeated attempts to obtain such literature I remained in the same predica- ment until I returned to England in 1899.
R E S E A R C H E S O N M A L A R I A 39 pared for the microscope), and was known to occur in from about 10 to 30
minutes after the blood is drawn from the patient; but it was now necessary to follow it in the mosquito. The insects were killed from one minute to sev- eral hours after feeding, the stomach being then extracted and its contents ex- amined in the fresh state. I was obliged to invent the technique for myself; and my first successful dissection was made on the 13th May. Within a few weeks I made a fairly complete study of the subject and ascertained an encouraging fact. In vitro, doubtless owing to the unnatural conditions, only about five per cent of the crescents give issue to the motile filaments; but I now found that in the mosquito’s stomach something like sixty per cent of them do so. It was also noted that the preliminary stages of the process, namely the swelling up and rounding of the crescents, were much more constantly seen in the blood ingested by the insect than in vitro. This was of importance because it showed at least that the insect’s stomach is a more favourable locus for the process than an ordinary specimen of blood is. I observed also that a considerable percent- age of the crescents (about one-third) never produced motile filaments at all,* even after the lapse of several hours and within the insects; and I noticed that those which refused to emit them had a slightly different appearance to the others. At the time I thought that they were parasites which had been killed in some manner during ingestion; but when I repeated the experiments next year I saw cause to doubt this, and felt some difficulty in explaining why all the crescents did not emit filaments as they should have done according to Manson’s hypothesis regarding their nature. A description of these first results was written in June, but was not published until the end of the year 24 .
methods devised. The fact, then, was established that the gametocytes are not immediately killed in the mosquito’s stomach (as might well have happened), but indeed emit their motile filaments more readily there than in vitro. It was necessary now to seek the destination of the latter in the insect’s tissues; and here the true magnitude of the task to which I had set myself became manifest. Manson had been able to follow the migra- tions of the filaria embryos with comparative ease because they are large or- ganisms readily distinguishable from the fluids or tissues which surround them. But the motile filaments are exceedingly delicate bodies, the move- ments of which are very difficult to follow even with the highest powers of a * It was easy to distinguish those which had produced the filaments by their collapsed condition afterwards. 40 1 9 0 2 R . R O SS good microscope and in the clear spaces of an ordinary preparation of blood. But the blood in a mosquito’s stomach shortly becomes a thick grumous mass in which it is impossible even to see the filaments unless they are in active movement. Moreover, even this assistance was denied me; for I speedily ascer- tained that within a few minutes of their escape they seemed to lose their movements. At least they constantly disappeared as if by magic; and in spite of all artifices I failed to ascertain what had become of them.* In fact to trace them further, to follow the migrations of these well-nigh invisible bodies through the masses of cells of which so large an animal as a mosquito is com- posed was indeed an impossible task with the means which I possessed. Hence, though Manson then and later constantly advised me in his letters to adhere to the plan of following the motile filaments, I determined to abandon the quest and to emply other methods; and he himself failed to obtain any results with the insects which I sent to him from time to time. It was most fortunate that I came to this decision so early, because events have proved that the motile filaments migrate nowhere, and do not enter the mosquito’s tissues at all. The first method which I now adopted and which ultimately led to success was the following. By hypothesis, the motile filaments after reaching the par- ticular cells of the mosquito for which we thought they were destined, should grow in them into some unknown but larger form. It was impossible to pre- dict what this form would be. Manson conjectured that it would very likely be some intracellular form similar to the intracorpuscular stages of the organ- ism in the human blood - fixed perhaps in the stomach cells or blood cells of the insect. Personally, however, while I thought this view possible, I had no full faith in it. It seemed to me that the mosquito stage of the parasite might be anything, so long as it was of a protozoal character. The protean changes of many of the parasitic worms warned us that nature was capable of ordering any extraordinary transformations in the interest of parasites; and as no case was yet known of a protozoon capable of wandering from one species of host to another, I had no guide as to what might happen with the organisms which I was studyin g, and conjectured that for all we could say, the motile filaments might develop into almost any form - amoe- boid, coccidiform, gregarinoid, or even infusorial, small or large. What was nearly certain, however, was that they were likely to grow in size after a few * It might now be possible, though still difficult, to follow them by staining them either in dehaemoglobinized blood or in section; but the Romanowsky reaction was not known Yüklə 2,27 Mb. Dostları ilə paylaş:
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