# Popular Science Monthly/Volume 59/June 1901/The Malaria-Germ and Allied Forms of Sporozoa

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* THE MALARIA-GERM AND ALLIED FORMS OF SPOROZOA.
By Dr. GARY N. CALKINS,
COLUMBIA UNIVERSITY.
THE group of animal parasites to which the malaria-causing organism belongs is relatively unimportant when compared with the bacteria, a group of plant parasites, including the causes of most zymotic diseases in man—typhoid, cholera, diphtheria, scarlet fever, tuberculosis and the like, as well as beneficial forms which aid man in various ways. Nevertheless, it is a group of considerable economic importance, about which little is known outside of scientific circles. The name Sporozoa suggests, to the average reader, no disquieting apprehension of physical pain or of financial loss, yet this class of primitive, unicellular animals includes, besides the malaria-causing blood parasites, forms which, like the silkworm parasite (Glugea bombycis), have cost communities untold millions of dollars. In connection with the losses due to one of these silkworm epidemics, Huxley writes in 18.0:
Analogous epidemics, which may be traced to Sporozoa, are liable to break out at any time among other animals having commercial value. Thus 'Texas fever,' a cattle disease due to a sporozoan blood parasite (Piroplasma bigeminum), occasions great loss to cattle breeders. Muscle parasites, belonging to the same class, cause trichinosis-like diseases in hogs, cows, cats, dogs and other domestic animals; while in fish they occasion great loss to fish-culturists through epidemics. Other parasites in the same class are the causes of disease in horses, sheep, goats, etc.
The Sporozoa are comparatively harmless to man personally, but, unlike some bacteria, they are never beneficial in any sense. Invariably parasites, the diseases which they induce are confined mainly to the lower animals, but so widely are they distributed that no type of animals is free from them altogether. One significant feature about the ​Sporozoa is that, notwithstanding the many kinds and the wide distribution in all sorts of hosts, the life history of the parasites invariably conforms to the same type, a fact which has recently been used to good advantage in working out the development of the malaria organism.
Like all the unicellular animals, or Protozoa, the Sporozoa are minute bits of protoplasm provided with a membrane and a specialized spherical portion of the inner protoplasm called the nucleus. Unlike the other Protozoa, they are entirely devoid of motile organs and are, in consequence, quiescent. In classifying them, advantage has been taken of the different modes in which they form spores, or germs, by which they are reproduced. In some, known as the Telosporidia, all the protoplasm of the parasite is used to form the spores, and the parent cell dies or disappears with each sporulation, which thus represents the end of the individual parasite. The individuals of the second group, known as the Neosporidia, form spores, without using all the protoplasm, and continue to live after each sporulation. This group comprises the less-known forms of Sporozoa, and is of considerable economic importance as the cause of epidemics among silkworms, brook trout and other fish, etc.
The Telosporidia are further divided according to the mode of life. Some of them, known as the Gregarinida, live in cavities of the body of many forms of invertebrates, but rarely in vertebrates; others, the Coccidia, live in epithelial cells lining the cavities of both invertebrate and vertebrate hosts. It may be remarked, parenthetically, that the cause of cancerous growths in man is claimed by many to be organisms belonging to this group of Sporozoa. The question remains in considerable doubt, however, and, despite the great mass of literature, no positive results have appeared. The last group finally of the Telosporidia is the Hæmosporidia, comprising parasites which, like the malaria-organism—Plasmodium malariæ—live in blood corpuscles of vertebrates.
All these different types of Telosporidia begin life as minute germs called sporozoites, which make their way into the new host through the intestine, being taken in with the food. The life history, after this ingestion, follows slightly modified paths in the different types, and, for purposes of comparison, I will describe these processes in the gregarine, the coccidium and in the hæmospore Plasmodium malariæ, thus representing each of the subdivisions of the Telosporidia.
The sea-squirt, or Tunicate, Ciona intestinalis, is the host of a gregarine Monocystis ascidiæ, which is so widely distributed that it is almost impossible to find a Ciona without them. The complet e life history of the parasite has been fully worked out by Prof. M. Siedlecki, ​of Cracow University, Russia, whose results form the basis of the following account:
The description of the life cycle may begin with the sporozoite, or youngest form, of the gregarine parasite. This is a small, elongate germ which makes its way through the fluids of the digestive tract of Ciona to the epithelial cells which line that canal, (l'ig. 1, A.) The sporozoite penetrates one of these cells and begins to grow at the expense of the cell contents, until, finally, too large for the cell host, it breaks the cell wall and falls into the lumen of the digestive tract, where it soon attains its full size. (Fig. 1, B. C. D.) It is now a comparatively large, sac-like cell, swollen at one end, and with a distinct nucleus. (Fig. 1, C.) After a longer or shorter period, not definitely determined, two adult forms come together and pour out a sticky, fluid substance, which soon hardens to form a common, firm covering, or cyst. (Fig. 1, E.) Each nucleus then begins to divide, and, after a multitude of daughter-nuclei have arisen, the protoplasm of the cell breaks np into as many parts as there are nuclei (Fig. 1, F. G. H.). These small protoplasmic parts (gametes) then wander out of the parent membranes and ultimately fuse, two by two, while still remaining in the original cyst wall (Fig. 1, I. J.). After the fusion, the nucleus and protoplasm in each double mass divides into eight parts, and a firm, enveloping membrane is secreted about them. This spore-membrane ultimately becomes impregnated with calcareous material, which thus forms a firm and resisting capsule for the eight germs within. Each germ is a sporozoite similar to the one which began the life cycle.
During the process of sporozoite-formation, the parasite is passed out with the fæces to the exterior. Here the original cyst ultimately bursts and liberates the multitude of spores with their contained sporozoites. The latter are well protected, however, by their calcareous shells, and do not suffer from the sea water or from drying. The spores may be finally taken into the digestive tract with food, and with this the opportunity for a renewed cycle is presented. The acids of the digestive fluids dissolve the calcareous coverings, and the eight sporozoites in each spore are liberated. The sporozoites again penetrate the epithelial cells, grow to maturity and repeat the process indefinitely.
In Coccidium, a parasite of some of the insects, the life history as worked out by Dr. F. Schaudinn differs in one or two important points from that of the gregarine.
Sporozoites are formed as in the previous case, and these work their way in a similar manner into the cells lining the digestive tract (Fig 3, a). Unlike the gregarine, the main period of their life is passed in these cells, and they drop into the lumen of the intestine only when they ​are ready to form spores. The nucleus divides repeatedly, and a great number of buds are formed around the daughter nuclei (Fig. 2, b, c). These buds elongate from the periphery of the parent organism and radiate from it, like the spines of a sea urchin. When fully developed, the spores, or, as they are technically known, the merozoites, drop off the parent cell and work their way through the fluids of the digestive tract until they come to the cells lining it, and then, like the sporozoites, they penetrate the cells, grow at their expense, and again reproduce spores as before (Fig 2, a to c). This process thus tends to spread the disease among the cells of the digestive tract in the one host, and it will be observed that the reproductive process is not accompanied by the union of two gametes, as in the case of Monocystis. Coccidium is thus distinguished from the latter in having a method of asexual multiplication leading to auto-infection. This process, however, cannot continue indefinitely, and, after five or six days, a method of sexual multiplication supervenes. The preliminary stages of this process do not differ from the formation of the merozoites, and similar buds are formed which break off' and penetrate the epithelial cells as before. The further history, however, differs markedly from that of the merozoite. Some of the resulting parasites give rise to immense numbers of minute, active, thread-like buds, the microgametes, which radiate from the parent cell like the merozoites (h——j). Others do not form buds at all, but merely enlarge until they are as large, or larger than, the ordinary full-grown parasites (d——f). One of the small forms then fuses with a large form, in conjunction; and the result, or copula, secretes a firm cyst about itself, and then divides into spores (2, g). Each spore then secretes about itself a second coating which becomes impregnated with calcareous matter, and, within this cyst, the cell divides into a small n,umber of sporozoites (k). In this condition the primary cysts are emptied to the outside, where they are ultimately taken up by some new host in whose digestive tract the cysts are dissolved and the sporozoites liberated to renew the cycle (Fig. 2, l).
It thus appears that, in Coccidium, the life cycle is more complicated than in the gregarine, in having a period of asexual reproduction by which auto-infection is accomplished, alternating with a period of sexual multiplication during which the parasite is carried from one host to another. Coccidium differs further from Monocystis in that the conjugating gametes are sexually differentiated, the small, active one, or microgamete, functions as the male cell, and the larger, quiescent one, or macrogamete, as the female or egg cell, while in the gregarine, on the other hand, the conjugating gametes are of equal size.
We may now consider the somewhat more complicated life cycle of the malaria organism. The process of spore-formation of this ​parasite, in the blood cells of the human host, was correctly made out in 1888 by two Italian naturalists, Marchiafava and Celli, who showed that the young parasite, in a red blood corpuscle, is a minute granule in which no structure could be made out. The granule grows, however, at the expense of the hæmaglobin of the corpuscle, and ultimately forms spores (Fig. 3, a—f). During the life of the parent organism, the products of growth are stored up in the parasite in the form of fine granules.
Minute germs, sporozoites (A), enter epithelial cells lining the digestive tract of a tunicate. Here they grow to a large size (B, C), ultimately breaking through a cell-membrane and falling into the lumen of the digestive tract (D). After some time in this adult condition, two individuals come together (E). The nuclei divide repeatedly (F, G), and minute gametes are ultimately formed (H, I). The gametes then fuse, two by two (I, J), forming the spores. The two nuclei also fuse (K), and the joint nucleus then divides three times in succession (L, M, N), forming right daughter-nuclei, which become the nuclei of eight germs or sporozoites (0). The sporozoites are inclosed in small calcareous capsules which, in a new host, are dissolved by the acids of the digestive fluids, thus setting free the sporozoites (A).
These, known as melanin granules, are left in the center of the parent organism when the spores are formed, but at this period the blood corpuscle, in which the sporulation occurs, disintegrates, and so liberates the spores and the melanin in the blood plasm. Like the merozoites of Coccidia, these spores make their way to new corpuscles, which they ​enter, and in which they repeat the cycle, thus bringing about autoinfection.
Another Italian naturalist, Golgi, in 1889, showed that the spore formation of the parasites and the well-known pyrexial attacks on the part of the patient occur at the same time, and the phenomena were interpreted as cause and effect. The direct cause of the attack was then found to be the liberation into the blood plasm of the melanin
granules, which, acting like a poison, throw the entire system into disorder. In different types of malaria, the attacks sometimes occur every 72 hours, sometimes every 48 hours, and in some cases at irregular intervals. These different effects are produced by slightly different forms of the malaria organism. One form, known as Plasmodium malariæ, sporulates every 72 hours; another, Plasmodium ​vivax, every 48 hours. Another form, giving rise to a more malignant type of malaria, is Laverania malariæ, which probably sporulates every 48 hours. Tn some types of the disease it is supposed that two or more
species may be present at the same time, and, sporulating at different intervals, may give rise to irregular attacks.
Until 1896, the life history of the parasite, as outlined above, was regarded as incomplete because of the perplexing form discovered in ​the blood of malaria patients by Professor Danilewsky, in 1891. This form differed from the ordinary parasites in having many fine, flagelliform appendages, which, breaking away from the parent, would swim about freely in the surrounding fluid (Fig. 3, p). Danilewsky regarded this form as an independent blood parasite, and gave to it the name Polymitus. In a sense, Polymitus has been the key to the life history of the malaria organism, and its history has been the history of the further discoveries upon malaria. In France, Laveran regarded Danilewsky's discovery as indicating some stage in the cycle of Plasmodium malariæ, and not as an independent organism, while Labbé considered Polymitus a degenerate condition of the ordinary parasites and without further significance. The English specialist on tropical diseases. Dr. Manson, found that the malaria parasites, when exposed with the blood to the cooler air, very soon assume the Polymitus form, which he regarded as the extra-corporeal form assumed by the malaria-' organism, for, he argued, the wide distribution of malaria, the spread from individual to individual, can be explained, since the disease is not contagious, only by the assumption of germs outside of the body. Furthermore, he suggested that these germs might be carried from person to person, by insects, such as the mosquito. In the same year, Laveran made an identical suggestion quite independently of Manson. It was not altogether novel, however, with either of these investigators; thus a certain mosquito in central Africa is known to the natives as the fever organism, while the same idea was represented in Theobald Smith's discovery (1893) of the tick as the agent in the transmission of the 'Texas fever' of cattle.
The first positive results on the significance of the Polymitus form were obtained by MacCallum, in Washington, in 1897-'98. A similar Polymitus form is developed by the malaria-organism of birds (Halteridium), and, in the blood of diseased crows, MacCallum observed the filamentous motile bodies of the Polymitus form, break away from the central mass, and unite with an ordinary parasite. The result of the conjugation was a copula with an independent motion, by which it made its way through the surrounding fluids. The later history of the copula was not followed; similar observations were made, by the same observer, upon living malaria-organisms of man, and the Polymitus 'flagella' were seen to unite with larger forms of the parasite.
In the meantime, Major Ross, in India, was working out the mosquito hypothesis of Manson and Laveran, and succeeded in placing that theory upon a very substantial basis. He found black pigment granules, in the intestine and epithelial cells of the mosquito, which were identified as melanin granules of the blood parasite. It was also found, by Ross, that only certain kinds of mosquitoes were selected by the malaria parasites, viz.: various species of the genus Anopheles ​by the human parasite, while species of the genus Culex were selected by the malaria parasites of birds. The full history, finally, has been worked out in complete detail during the last two years, by Ross again, and by Grassi, in Italy, and both observers reached quite independent, but identical, results. Briefly summarizing these results, the full life history of Plasmodium malariæ may be given as follows:
The early form of the parasite, which corresponds with the sporozoites of the gregarine and of Coccidium, penetrates a red blood corpuscle, grows to adult size, and then forms spores (Fig. 3, a——b). These correspond exactly with the merozoites of the Coccidia, and, like them, lead to auto-infection. At this point there is a gap in the evidence, for it is not known how long this asexual method of increase may continue; as shown above in the case of Coccidium, the sporeforming period continues for five or six days, when a period of conjugation supervenes. It may be stated here, parenthetically, that in all Protozoa, so far as known, a period of conjugation is necessary at some time during the life cycle, and without such conjugation, the organisms, which are reproduced asexually, finally decrease in size and show other signs of degeneration, ultimately resulting in death of the race (see results of Bütschli, Maupas, Hertwig, etc., upon degenerating Protozoa).
This is of considerable moment in the question of malaria, for, if the malaria-organism conforms to other Protozoa, there must come a time when this asexual sporulation will cease in any given set of individuals, and a period of conjugation must supeivene to give renewed vigor to the parasites. So far as known at the present time, this conjugation takes place only in the digestive tract of the mosquito. That it does actually take place, is undeniable from the observations of MacCallum, Ross, Grassi and others, and the conjugants again as in Coccidium, are: a small, motile, microgamete, or inale cell (one of the 'flagella' of the Polymitus form); and a larger macrogamete, or female, cell (Fig. 3, p——v). Their union, observed by Ross and Grassi, takes place in the stomach of Anopheles, and the copula then makes its way into, and through, the epithelial cells lining the stomach, and finally rests against the tissue which lines the body cavity. Here it grows to a relatively large size (Fig. 3, w——zz), and, when mature, its nucleus divides as in the gregarine or in Coccidium, to form a number of spores. Each of these develops a number of germs, or sporozoites, but, unlike the sporozoites of the previously described Sporozoa, these germs have no protective capsule about them, and, when the parent cyst ultimately bursts, they are liberated directly into the body cavity of the mosquito (Fig. 3, A, B). Here, in the fluids of the body cavity, they are carried to all parts of the organism, and finally reach the anterior region of the thorax, where the ​salivary glands of the insect are located. They work their way through the cell wall of this gland, into the gland cells, from which they are drawn with the secretion and are finally poured into the lumen of the gland (Fig. 3, C). When the proboscis of the mosquito is inserted into a human host, and the salivary secretion is poured out, the sporozoites pass with it into the blood, and thus effect infection of a new host. Provided with their new potential of vitality resulting from conjugation, the young sporozoites grow and multiply in the blood corpuscles until they are numerous enough to cause the well-known symptoms of malaria. Coming from the same brood, so to speak, they have a similar rate of growth and multiplication, and so liberate their melanin granules throughout the blood system of their human host, at approximately the same time.
The question is frequently asked: Is the mosquito the only agent in the transmission of malaria? and when this is answered by the somewhat modified affirmative, 'Yes, so far as we know,' it is usually followed by the query: 'Why does malaria follow bad drainage, the digging of sewers, laying of gas pipes, etc.?' This question may be answered in two ways: First, it must be shown that these so-called malarial fevers, which accompany such conditions, are in reality true malaria; it is quite possible that hasty diagnosis in many cases gives a wrong impression of the prevalence of this disease. Second, it is conceivable that sporozoites may be carried in the blood, as typhoid is said to be frequently carried in the digestive tract, without causing symptoms of the disease until the natural resistance of the host is weakened by decreased vitality, which may be brought about by bad air, or by other means.
It is quite possible that some other means of transmission than the mosquito exists. The flea, for example, and other insects that prey on man must be examined with this end in view. There is no reason to believe that the sporozoites can be liberated in water, or suspended in the dust of the air, and live, for, of all Sporozoa, the blood-infesting forms are not protected against an external life. Thus we have seen that the sporozoites of the gregarine or of Coccidium are incased in a firm, calcareous shell, which protects them from drying and from other dangers that might be encountered. With the malaria-organism there is no such coating; the sporozoites are at all times naked bits of protoplasm, which soon dry up and die, when exposed to the air or placed in water. This fact also refutes the argument made by Bignami and others that the parasites are transferred directly from one individual to another by sticking to the proboscis. It is probable that the mosquito is the original, or primary, host of the malaria-organism, and that man and birds are secondary hosts, from which the parasites return to the primary one for the vital function of conjugation.