Popular Science Monthly/Volume 50/March 1897/The Malarial Parasite and Other Pathogenic Protozoa
By GEORGE M. STERNBERG, M. D., LL. D.,
SURGEON GENERAL, UNITED STATES ARMY.
GENTLEMEN: My presidential address last year had for its subject The Practical Results of Bacteriological Researches, and at its conclusion I showed you upon the screen photomicrographs of the principal pathogenic bacteria, including the micrococcus of pneumonia; the micrococci concerned in the production of boils, abscesses, wound infection, puerperal fever, erysipelas, etc.; the bacilli of tuberculosis, of diphtheria, of typhoid fever, of glanders, of anthrax, of influenza, of tetanus, of leprosy; the spirillum of relapsing fever, the spirillum of Asiatic cholera, and various other pathogenic bacteria. These micro-organisms are now generally recognized as belonging to the vegetable kingdom, and as a specialist in this department of scientific investigation your president may perhaps be considered a botanist in a small way. But the Biological Society includes among its members many distinguished specialists in that branch of natural history which relates to the animal kingdom, and I think it due to the zoologists to show that the botanists have no monopoly of mischief-making micro-organisms. In speaking of the pathogenic protozoa I shall devote special attention to the hæmatozoon which is now recognized as the specific cause of the malarial fevers; and in consideration of the importance of this blood parasite from a sanitary point of view, as well as of the interest which attaches to it from a biological standpoint, I shall occupy a little time in giving you an account of its discovery and the grounds upon which it is accepted by well-informed pathologists as the specific infectious agent in the class of fevers referred to.
The malarial parasite was discovered in 1880 by Laveran, a surgeon in the French army, at that time stationed in Algeria, but now a professor in the military school at Val de Grâce.
In my work on Malaria and Malarial Diseases, published in 1884, I refer to Laveran's alleged discovery as follows:
"According to this observer, there are found in the blood of patients attacked with malarial fever pigmented parasitic elements which present themselves under three principal aspects. This parasite is said to be a kind of animalcule which exists at first in an encysted state. In the blood these organisms present themselves as motionless, cylindrical, curved bodies, which are pointed and transparent and have a pigment spot; also as spherical bodies, about the diameter of a red blood-corpuscle, showing active movements and containing in their interior numerous pigment granules. The movements of these bodies are due to the action of elongated filaments attached to their circumference—flagella. A third form in which these parasites present themselves in the blood is as motionless, spherical, or irregular-shaped bodies containing dark-red, rounded pigment grains. These bodies have no nucleus, and do not stain with carmine; they appear to be the ultimate stage of development of the above.
"The blood also contains free pigment granules, pigmented leucocytes, and vacuolated red corpuscles which contain pigment granules.
"These parasitic elements have only been found in the blood of persons sick with malarial fever, and they disappear when quinine is administered.
"They are of the same nature as the pigmented bodies which exist in great numbers in the vessels and organs of patients dead with pernicious fever, which have been described as melanotic leucocytes. Laveran, at the time his report was published, had found these bodies in one hundred and eighty out of one hundred and ninety-two persons examined in Algeria and in Tunis who were affected with various symptoms of malarial poisoning.
"The presence of the parasite described by Laveran in the blood of persons suffering from malarial diseases is confirmed by Richard, who has studied the subject at Philippeville, France, where malarial diseases abound. This author has invariably found the parasite of Laveran in the blood of malarial-fever patients, and has never seen it in the blood of persons suffering from other diseases. He finds that its special habitat in the blood is the red corpuscles, in which it develops and which it leaves when it has reached maturity. During the attack of fever many blood globules are seen which possess a small, perfectly round spot. Otherwise they preserve their normal appearance; they are simply, so to speak, stung (piqués). Beside these globules are others in which the evolution of the microbe is more advanced. The clear spot is larger and is surrounded by fine black granules. The surrounding hæmoglobin forms a ring which decreases as the parasite augments in volume. After a time only a narrow, colorless zone remains at the margin. This corresponds with the body No. 2 of Laveran, ‘having about the dimensions of a red corpuscle and inclosing an elegant collarette of black granules.’ This collarette is the microbe which has arrived at its perfect state, and which is provided with one or several slender prolongations, measuring twenty-five micromillimetres or more in length. Richard has several times seen the fully developed parasite emerge from its ‘shell’—the remnant of the invaded red corpuscle—to which it may remain attached, and which can only be seen with great difficulty. Sometimes only the motile filaments penetrate the envelope in which the body of the parasite remains inclosed. In both cases the filament is seen to undergo active movements, and when its extremity is caught in the fibrinous reticulum the body itself oscillates. This movement may last for an hour. Usually, however, no movement is observed, and the corpuscles containing very small parasites never move. The infected corpuscles become disorganized, the pigmentary collarette is broken down, and a grayish mass inclosing some black granules remains. The pigment granules when set free are rapidly picked up by the leucocytes; the melaniferous leucocytes are therefore epiphenomena."
Commenting upon the observations of Laveran and Richard, I say, in the work just referred to:
"We can not doubt that a true account has been given of what the observers believe they have seen. But there is a wide field for doubt as to the deductions made from the various observations recorded; for in microscopical studies of the blood made with high powers there is a great liability to error and to misinterpretation of what is seen. We may question, for example, whether the belief of Laveran and Richard that the appearances noted by them are due to parasitic invasion of the blood-corpuscles is well founded, without calling in question the accuracy of their observations."
At the time this was written pathologists generally were not disposed to attach much importance to the alleged discovery of Laveran, and this was especially true in Germany and in those countries in which physicians were in the habit of awaiting the verdict of German bacteriologists before accepting anew "disease germ." One reason for this failure to give proper consideration to the discovery of Laveran was the fact that there was a rival in the field. A year before the publication of Laveran's paper, giving an account of his observations, the distinguished German pathologist Klebs, in association with the prominent Italian physician Tommasi-Crudeli, had announced the discovery of a bacillus which they believed to be the cause of the malarial fevers, and which they named Bacillus malariœ. Their experimental investigation was made in Rome with material obtained from the malarious marshes in the vicinity of that city.
The evidence offered in the original memoir of Klebs and Tommasi-Crudeli in favor of the view that the bacillus described by them is the cause of malarial fevers in man, is derived from experiments made upon rabbits, in which culture fluids containing the bacillus in question, and washings from malarious soils, were injected subcutaneously.
In my work on Malaria and Malarial Diseases, already referred to (published in 1884), I say:
"The importance of this alleged discovery induced the National Board of Health, soon after the publication of the first report published by Klebs and Tommasi-Crudeli, to undertake control experiments in a recognized malarial locality in this country. The writer, who had established a laboratory in New Orleans for the purpose of studying the micro-organisms present in the atmosphere of that city, was therefore instructed to repeat the experiments of Klebs and Tommasi-Crudeli, and during the autumn of 1880 devoted a considerable portion of his time to this investigation. The results obtained were not favorable to the view that the fever produced in rabbits by the injection beneath their skin of infusions of swamp mud, etc., was a truly malarial fever; and, for reasons stated, the conclusion was reached that the evidence offered by Klebs and Tommasi-Crudeli in their first report, which alone had been published at this time, was unsatisfactory. (The full report of these investigations is given in Supplement No. 14, National Board of Health Bulletin, published in Washington, D. C., July 23, 1881.)"
Referring to subsequent observations, I remark:
"Since the publication of the report above referred to the belief that the Bacillus malariœ, is the true cause of malarial fevers has received considerable support from observations made in Rome, under the direction of Tommasi-Crudeli, by Marchiafava, Cuboni, Peroncito, Ceri, and others.
"We do not feel prepared to estimate the value of this evidence in detail, but will, in a general way, give our reasons for considering it in a spirit of scientific skepticism, and for demanding substantial confirmation from other parts of the world where malarial fevers prevail, and especially in our own country, where malaria is so well known by its effects, and where the Bacillus malariœ. should be easily found if it is constantly present in the blood during the cold stage of intermittents, as has been claimed by some of the Roman observers. . . .
"The writer's observations lead him to be very cautious in accepting evidence relating to the discovery of organisms in the blood, when these are few in number and require diligent search for their demonstration; for the possibilities of accidental contamination or of mistake in observation are very great. . . .
"The writer has many times examined carefully the blood of malarial-fever patients with a one-eighteenth-inch oil-immersion objective (of Zeiss), and has not been successful in finding either rods or spores. But few of these examinations have, however, been made during the chill, and the blood has not been drawn directly from the spleen; these observations are therefore to be considered as incomplete, and, if opportunity offers, will be supplemented by more extended microscopical researches."
Notwithstanding this adverse criticism, based upon an experimental research made for the purpose of confirming the alleged discovery, if it should prove to have a scientific foundation, the Bacillus malariœ was pretty generally regarded by physicians in this country and in England as being the veritable cause of malarial fevers, and for several years it was frequently mentioned in medical journals and even in standard text-books of medicine as one of the demonstrated disease germs. But truth is mighty, and in the end must prevail. To-day no one speaks of the Bacillus malariœ of Klebs and Tommasi-Crudeli except to refer to it as one of the pseudo-discoveries, which for a time passed current, like a counterfeit coin, but which was detected and thrown aside when subjected to approved scientific tests.
The first confirmation in this country of Laveran's discovery of amœboid parasites in the blood of malarial-fever cases was made by the present writer in the pathological laboratory of the Johns Hopkins Hospital in March, 1886. In May, 1885, I had visited Rome as a delegate to the International Sanitary Conference convened in that city under the auspices of the Italian Government, and while there I visited the Santo Spirito Hospital for the purpose of witnessing a demonstration, by Drs. Marchiafava and Celli, of that city, of the presence of the Plasmodium malariœ in the blood of persons suffering from intermittent fever. Blood was drawn from the finger during the febrile attack and from individuals to whom quinine had not been administered. The demonstration was entirely satisfactory, and no doubt was left in my mind that I saw living parasitic micro-organisms in the interior of red blood-corpuscles obtained from the circulation of malarial-fever patients. The motions were quite slow, and were manifested by a gradual change of outline rather than by visible movement. After a period of amœboid activity of greater or less duration, the body again assumed an oval or spherical form and remained quiescent for a time. While in this form it was easily recognized, as the spherical shape caused the light passing through it to be refracted and gave the impression of a body having a dark contour and a central vacuole; but when it was flattened out and undergoing amœboid changes in form, it was necessary to focus very carefully and to have a good illumination in order to see it. The objective used was a Zeiss's one-twelfth-inch homogeneous oil immersion.
The changes in form which a single plasmodium, included in a red blood-corpuscle, was observed by Marchiafava and Celli to undergo within a period of twenty minutes I shall show yon upon the screen at the close of my address (see Fig. 1).
The confirmation of Laveran's discovery, as already stated, was first made by Richard in a communication to the French Academy of Sciences (February 20, 1882), then by Marchiafava and Celli
Fig. 1.—Figures 1 to 22 represent the changes in form which a single plasmodium, included in a red blood-corpuscle, was observed to undergo within a period of twenty minutes. Figures 23 to 27, 29, and 30 show other forms assumed by plasmodia, some with and some without pigment. Figure 28 shows a motionless plasmodium emerging from a red blood-corpuscle; the blood was taken after the paroxysm of fever and administration of quinine. (Marchiafava and Celli.)
(1883), and subsequently by Councilman and by Osler in this country, by Golgi in Italy, by Manson of England, and by many other competent microscopists in nearly all parts of the world where malarial diseases prevail.
The intracorpuscular development of the plasmodium has been traced by Golgi and others, and results in the formation of a number of bodies arranged in the form of a rosette, with a mass of pigment granules at the center (see Fig. 2). The growth of the plasmodium seems to be at the expense of the hæmoglobin of
Fig. 2.—Intracorpuscular Development of the Parasite of Tertian Intermittent Fever: 1, 2, young hyaline forms; 3, 4, more advanced pigmented forms; 5, fully grown parasite; 6, 7, 8, 9, segmentation and production of free spores (10).
the infected red blood-corpuscle, and the pigment granules are probably to be regarded as an excrementitious product. The segments of the rosette, which represents the final stage of intracorpuscular development, are finally set free by a breaking down of the remains of the corpuscle, and are supposed to correspond with the elementary body which invaded the corpuscle in the first instance. The periodicity of this class of fevers is believed to depend upon the fact that a certain time is required for the intracorpuscular development of the plasmodium, and that successive crops of these elementary bodies are set free at regular intervals. We have also in the blood of malarial-fever patients certain pigmented bodies which are believed to represent different stages in the development of the same parasite, or of a nearly related parasite, which is concerned in the ætiology of a different type of malarial fever. The form most frequently encountered is associated with the so-called "æstivo-autumnal" malarial fevers which prevail in the vicinity of Rome and elsewhere. The bodies characteristic of this type of fever are crescentic in form and contain black pigment granules, usually centrally located (see Fig. 3). These crescentic bodies are not usually found in the blood of persons suffering from intermittent fever of the tertian or quartan type. Golgi, who has made very extended studies of the blood of malarial patients, asserts that each intermittent paroxysm is associated with the segmentation of a group of intracorpuscular organisms—that is to say, that the paroxysm corresponds with the ripening of a generation of the parasites. He also concludes from his observations that the number of parasites shown to be present by a microscopical examination of the blood corresponds, in a general way, with the severity of the attack. These observations have been confirmed by Osier, Antolisei, and others. Golgi has noted differences between the plasmodium as found in the tertian type of intermittent and that found in quartan fevers; and these differences are sufficiently marked to enable him to determine the type of fever by a blood examination. These observations have been confirmed by numerous competent pathologists in various parts of the world. When a daily paroxysm occurs, according to Golgi, this is due to the alternate development of two groups of quartan parasites. He has not been able to demonstrate a special parasite of quotidian fevers having a cycle of intracorpuscular development lasting twenty-four hours.
The differences between the evolution of the tertian and quartan parasite are summarized as follows by Laveran, from the data given by Golgi:
The tertian parasite completes its evolution in two days, the quartan in three days.
The amœboid movements of the tertian parasite are much more active than those of the quartan.
Fig. 3.—Crescentic and Flagellate Forms Characteristic of "Æstivo-Autumnal" Malarial Fevers.
The tertian parasites cause a rapid decoloration of the red blood-corpuscles invaded by them, while the corpuscles retain their characteristic color when invaded by the quartan parasite up to the time that they are almost destroyed as a result of its intracorpuscular development.
In the quartan type the red blood-corpuscles containing the parasite have a tendency to become smaller than normal corpuscles; in the tertian type they are usually larger.
In the tertian the protoplasm of the parasites is very transparent; in the quartan it is less so, and the outlines are better defined.
In the quartan the pigment is seen in the form of grains or rods of greater size than in the tertian.
Finally, the principal difference is found in the segmentation of the intracorpuscular elements. The number of spores resulting from segmentation is greater in the tertian parasite—usually more than twice as many.
That these parasitic protozoa are in truth the cause of the forms of malarial fever with which they are found to be associated can scarcely be doubted, in view of the facts that this association is a constant phenomenon, that the infected blood-corpuscles are destroyed by the parasite, that a rapid loss of red corpuscles is one of the most marked results of malarial infection, and that the parasites disappear when quinine is administered in suitable doses, thus accounting for the specific action of this drug. Finally, it has been shown by inoculation experiments that when blood containing the plasmodium is drawn from the circulation of a malarial-fever patient and injected beneath the skin of a healthy person there is a reproduction of the parasite in the blood of the inoculated individual, and, after a certain period of incubation, typical malarial paroxysms occur. Successful inoculation experiments have been made by Marchiafava and Celli, by Gualdi and Antolisei, by Bein, by Bacelli, by Di Mattel, and others.
The life-history of the malarial parasite outside of the bodies of infected individuals has not been traced. Thus far attempts to cultivate it in artificial media have failed. Nor has the presence of the plasmodium been demonstrated in water or mud taken from the marshy localities which are recognized as the source of malarial fevers, and which we therefore believe to be the normal habitat of this mischievous micro-organism. The facts relating to the seasonal and regional occurrence of malarial fevers sustain the view that they are caused by living organisms, the external development of which depends upon conditions relating to temperature, moisture, vegetable growth and decay. But among the vast number of micro-organisms of an infinite variety of species found in localities which are recognized as malarious, the little specks of protoplasm which—with a first-class oil-immersion objective, and by careful manipulation of the light—we recognize as parasitic invaders of human blood-corpuscles could scarcely be detected, and could not be differentiated from other sporelike bodies in the absence of methods for obtaining each species separately, in pure cultures.
Thayer and Hewetson, in their admirable monograph on the Malarial Fevers of Baltimore (1895), in which they give an account of six hundred and sixteen cases observed by themselves, have summarized their observations relating to the parasite as follows:
"We have distinguished three varieties of the malarial parasite:
"1. The tertian parasite.
"2. The quartan parasite.
"(1) The tertian parasite requires about forty-eight hours to accomplish its complete development, and is associated with relatively regular tertian paroxysms, lasting on an average between ten and twelve hours, associated, almost always, with the three classical stages—chill, fever, and sweating. Frequently, infection with two groups of tertian organisms gives rise to quotidian paroxysms; rarely, infection by multiple groups of organisms gives rise to more irregular, subcontinuous fevers.
"(2) The quartan parasite is an organism requiring about seventy-two hours for its complete development. It is rare in this climate, and is associated with a fever showing regular quartan paroxysms, similar in nature to those associated with the tertian organism. Infection by two groups of the parasite causes a double quartan fever (paroxysms on two days, intermission on the third). Infection with three groups of the parasite is associated with daily paroxysms.
"(3) The æstivo-autumnal parasite passes through a cycle of development, the exact length of which has not as yet been determined; it probably varies greatly, from twenty-four hours or under to forty-eight hours or more. But few stages of development of the parasite are found, ordinarily, in the peripheral circulation, the main seat of infection being, apparently, in the spleen, bone marrow, and other internal organs. Infection with this organism is associated with fevers varying greatly in their manifestations. There may be quotidian or tertian intermittent fever, or, more commonly, more or less continuous fever with irregular remissions. The individual paroxysms last, on an average, about twenty hours. The irregularities in temperature depend, probably, upon variations in the length of the cycle of development of the parasite, or upon infection with multiple groups of organisms.
"We have not been able to separate two distinct varieties of the æstivo-autumnal parasite, though we feel that more investigation is needed upon the subject.
"The cases of malaria in the spring and early summer are of the milder, more regularly intermittent varieties (tertian and quartan fever), the severe æestivo-autumnal infections beginning to appear only in the later summer, and reaching their maximum in September."
Manson has recently suggested that the mosquito is an intermediate host for the malarial plasmodium. We have an analogy for this in the part played by the mosquito in withdrawing embryo filariæ (Filaria sanguinis hominis) from the blood of infected individuals and returning them to the stagnant pools frequented by the insect. Manson says, in discussing this hypothesis in his Gulstonian Lectures (1896):
"We can readily understand how the mosquito-bred Plasmodium may be swallowed by a man in water, as so many disease germs are, and we can readily understand how it may be inhaled in dust. Mosquito-haunted pools dry up. The plasmodia in the larvæ and those that have been scattered about in the water, finding themselves stranded by the drought, and so placed in a condition unfavorable for development, pass into a resting stage, just as they do when by quinine or other means man is rendered temporarily unsuited for their active life. They may, probably do, become encysted, as so many of the protozoa do in similar circumstances. The dried sediment of the pool, blown about by winds and currents of air, is inhaled by man, and so the plasmodium may find its way back again to the host from whom its ancestors had, perhaps, started generations back."
This theory appears plausible, but we find it difficult to believe that man is essential for the completion of the life cycle of the plasmodium, for the most concentrated and deadly malarial emanations may be given off from marshy places which are far removed from the haunts of men. It may be, however, that the mosquito is an essential factor in the development of the plasmodium, and that man, instead of being a necessary intermediate host, only serves occasionally, and in a certain sense accidentally, as such. Perhaps other mammals or birds may serve the same purpose. It has frequently occurred to the writer that the malarial plasmodium, like other amœboid protozoa, may find its normal habitat, external to the bodies of its insect or animal hosts, upon the stems and leaves of water plants, rather than in the water itself. The fact that malarial fevers do not prevail in the vicinity of swamps when the marsh vegetation is submerged by high water is in favor of this view; as is also its apparent need of plenty of oxygen, which we infer from its active multiplication in the blood and its parasitic invasion of the red blood-corpuscles.
Possibly the mosquito is an intermediate host for the Plasmodium malariœ on a larger scale than Manson suggests. The natural food of this insect is the juices of plants, and, no doubt, a vast majority of them never have a chance to fill themselves with the rich red fluid from their human victims which they are so eager to substitute for their normal diet when opportunity offers. If, as we have suggested, the plasmodium abounds upon the stems and foliage of herbaceous plants in marshy localities, the mosquito would be very likely to pick it up in following its everyday method of gaining a livelihood.
Parasitic protozoa, closely resembling the malarial parasite, have been found in the blood of birds and of reptiles, and possibly one or more species of lower animals may serve as an "intermediate host" for the hæmatozoon under consideration. Laveran has given a drawing, in his work on Paludism, of a parasite found in the blood of the lark, which is evidently of the same family. The fact that a parasite may develop in the blood, or elsewhere, in one or more species of animals without giving rise to any evident symptoms of disease can not be taken as evidence that it is not pathogenic for man, or for some other animal. On the contrary, we have numerous instances which show that animals may have a natural or acquired immunity to the pathogenic action of parasitic micro-organisms which are deadly for other animals of the same or different species.
Texas fever, an infectious disease of cattle which prevails as an endemic disease in certain regions in the southern portion of the United States, has been shown, by the researches of Theobald Smith and other bacteriologists belonging to the Agricultural Department, to be due to a blood parasite belonging to the protozoa (Pyrosoma bigeminum of Smith). In this disease the tick has been shown to be the intermediate host of the parasite. The ticks which fall from infected animals give birth to a numerous progeny in the pastures frequented by them, and these young ticks attach themselves to other animals which subsequently feed in the same pastures and transmit to them the fatal infection.
The tsetse fly disease of Africa has recently been shown by the researches of Bruce to be due to a flagellate infusorium which is found in the blood of infected animals. This disease is fatal to the ox, the horse, the dog, the sheep, and the ass, but not to the indigenous wild animals in the region infested by the tsetse fly. The researches of Bruce indicate that the fly acts as a carrier of the parasite from diseased to healthy animals. He has shown by experiment that after feeding on the blood of an infected animal the tsetse fly can communicate the disease to a healthy animal by its bite. After a short period of incubation the hæmatozoa appear in the blood concurrently with the development of fever, and followed by rapidly progressive anæmia, dropsy, and death.
The so-called "surra disease," which prevails in certain portions of India, is believed to be due to a similar parasitic protozoan (Trypanosma Evansi). According to Lingard, this infusorium exists as an innocuous parasite in the blood of rats in India. It is not pathogenic, or only feebly so, for the native ox of India, but gives rise to a fatal infectious disease in horses, dogs, and camels.
Another pathogenic micro-organism belonging to the protozoa is the Amœba coli, which is found in great numbers in the large intestine in cases of tropical dysentery and also in liver abscess secondary to this disease.
The rapid progress of our knowledge of the bacteria has been due to the fact that satisfactory methods (staining) have been devised for detecting these minute micro-organisms in the blood and tissues of infected individuals and for cultivating them in artificial media. Unfortunately, these methods have only a limited utility when applied to investigations relating to the protozoa. The bacterial cell has considerable stability, owing to its cellulose envelope (cell wall), and it is readily stained by the aniline dyes. The protozoa, on the contrary, very readily undergo disintegration, and the more fluid protoplasm of these unicellular organisms is not so easy to demonstrate by the usual staining reagents. It has also been found very difficult, and in many cases quite impossible, to obtain pure cultures in artificial media. Again, the recognition of protozoa in the blood of infected animals by means of the microscope requires special skill in making preparations, in the management of the light, etc., and expert knowledge of the normal elements of the blood and of the changes they undergo as a result of various methods of preparation. This is illustrated by the fact that many persons, more or less familiar with the use of the microscope, have failed to discover the malarial parasite in blood which undoubtedly contained it, while others have evidently mistaken vacuoles in normal blood-corpuscles for the Plasmodium, the crenated red corpuscles for pigmented cells, and deformed corpuscles for malarial crescents.
Notwithstanding the painstaking researches which have been made during the past few years for the purpose of determining the nature of certain bodies which may be demonstrated by special staining methods in the cells of carcinomatous tumors, we are still uncertain as to the nature and ætiological import of these bodies. Some investigators believe them to be protozoa, and from their location infer that they are the specific ætiological agents in the development of malignant growths of this character. But, so far as we are informed, this view has not as yet received any very substantial support, and has not been accepted by the leading pathologists of the world.
The presence of amœboid micro-organisms in the contents of the pustules of variola and in vaccine lymph has been reported by several investigators: Guarnieri (1892), Monti (1894), Piana and Galli-Valerio (1894), L. Pfeiifer (1894), Clarke (1895), von Sicherer (1895), E. Pfeiffer (1895). Guarnieri in 1892 published a paper in which he claimed to have cultivated the amœboid micro-organism found by him in vaccine lymph by successive inoculations in the cornea of rabbits. E. Pfeiffer has since (1895) confirmed this observation, and has seen the parasite undergoing amœboid movements and in progress of multiplication by spontaneous fission. During the past two years investigations relating to the ætiology of vaccinia and variola have been made at the Army Medical Museum in Washington, by Major Walter Reed, surgeon United States Army. These investigations show that in vaccinated monkeys and in children an amœboid parasite makes its appearance in the blood on the sixth or seventh day after vaccination, and may be found during a period of from five to seven days, when it disappears. Reed has found the same parasite in the blood of patients with variola and in his own blood after an accidental vaccination in the finger. The parasites are not numerous. They are less than a third the diameter of a red blood-corpuscle, and may be observed to undergo amœboid movements in a drop of blood, properly mounted for microscopical examination, during a period of twenty-four hours or more. These amœboid bodies, like the malarial parasite, would be easily overlooked by one not an expert in blood examinations.
The presence of a ciliated amœboid micro-organism in the mucous secretion coughed up by children suffering from whooping-cough has recently been reported by Deichler and confirmed by Kourlow. This may prove to be the cause of the disease, but further researches will, of course, have to be made before this can be determined. In view of the extended investigations made during the past few years by competent bacteriologists, it seems probable that in most of those infectious diseases in which the specific infectious agent has not yet been discovered it belongs to some other class of micro-organisms than the bacteria; and it seems not improbable that some of them at least will prove to be due to infection by "germs" belonging to the class to which your attention has been invited in the present address—viz., the pathogenic protozoa.
M. Henri de Kerville describes fifteen yew trees in Normandy which are supposed to be a thousand years old and more; a number of oaks from three hundred to nine hundred years old; cedar trees from a hundred to a hundred and fifty; a hawthorn, two hundred; a pear tree, more than a hundred; a holly tree, a hundred or a hundred and fifty; and an American tulip tree, a hundred or a hundred and twenty years old.