Tropical Diseases/Appendix

by Patrick Manson

Appendix

SOME OF THE COMMONER PARASITIC PROTOZOA

OF VERTEBRATES AND INVERTEBRATES

RECENT discovery has clearly demonstrated the importance of the Protozoa as factors in the etiology of disease, especially of tropical disease.

Manifestly the subject is still in its infancy. Many as yet unknown species of blood protozoa will doubtless be added to the already considerable list of those attacking man and the lower animals. As regards man, it is doubtful if we have as yet completed the list even of the much-studied malaria parasites, much less determined their respective life histories and zoological relationships. Moreover, there are certain genera of blood protozoa which, although occurring in other mammals, have, so far, not been found, or, at all events, recognized in man, Babesia and Hæmogregarina for example. It is not unreasonable to conjecture that, although hitherto unrecognized, representatives of these genera do occur in man, and that ere long they will be discovered. The investigator must not lose sight of this probability.

The entire subject is one of the utmost importance to the medical man in the tropics, and, whether he would perfect himself in existing knowledge or seek to add to it, he should study it practically and from the standpoint of comparative pathology. I would therefore strongly recommend him to avail himself of the abundant opportunities he is sure to enjoy to study the protozoa not in man only but in other mammals, in birds, in fishes, and in reptiles. As a guide and help in such studies I append a brief summary, for which I am indebted to Dr. Philip Bahr, of our present knowledge of this important class of parasites, so far as it has a bearing on human pathology. As to classification, still a quœstio vexata, the one adopted in Prof. Minchin's " Introduction to the Protozoa " (1912) has been for the most part followed.

DEFINITION OF THE PROTOZOA

Protozoa are unicellular organisms possessing a nucleus distinct from the cytoplasm. They belong to the Protistenreich of Haeckel, a kingdom which unites the simplest and most primitive forms of life, and which may be considered equivalent in systemic value to the animal and vegetable kingdoms.

The protozoal cell constitutes an entire individual; it may exist singly, or combined in the form of cell colonies.

The body is a mass of protoplasm sometimes enclosed in a limiting envelope. Such organs as are present are also protoplasmic, and are used for the purposes of locomotion and capture of food. Metabolism is generally of the animal type.

Reproduction takes place by fission. Sexual reproduction generally occurs throughout the group, and, as compared with other Protista, the developmental cycle may be a very complicated one in which the same organism reappears in a totally different form at different periods of its development.

There are certain forms of development in the blood protozoa which it is necessary to define and for which certain terms are used. Their application, however, when loosely used has given rise to much confusion. The protozoa parasitic in blood of the vertebrate host undergo a vegetative or non-sexual multiplication, the process being then known as schizogony; but in order to ensure their further existence, and their passage from one vertebrate host (intermediary host)*^[1] to another, a further cycle in a cold-blooded invertebrate host (definitive host) is necessary, during which the sexual cycle or sporogony takes place.

In the vertebrate blood the young parasite is known as a schizont. The products of the asexual fission are known as merozoites; these merozoites at some period of their existence develop into forms known as sporonts, destined to pass into the alimentary tract of the inveitebrate host. The term trophozoite is applied to the young schizont or sporont which, is absorbing nourishment direct from the cell in which it is lodged.

Parthenogenesis is not recognized as occurring in the protozoa, but was supposed by Schaudinn to take place in the case of the malaria parasite and to be responsible for the relapses of that infection.

These sporonts, still in the blood of the vertebrate host, develop into sexually differentiated cells (gametocytes), which are quiescent until they are removed from the blood; when this occurs both male and female cells extrude two polar bodies preparatory to the sexual process, and are then known as gametes. The flagella or microgametes extruded by the male cell function as spermatozoa, enter and fertilize the female cell (macrogamete), thereafter known as a zygote.

After this act the zygote in some forms, especially the Plasmodidæ, becomes an elongated motile body (oökinete or travelling vermicule), which may remain free or become encysted (oöcyst). The oöcyst gives rise to secondary bodies in its interior (sporoblasts), which in turn develop into a number of minute rods known as sporozoites. Usually these sporozoites continue to enter the mouth parts of the insect, and by this route they once more gain the blood of the vertebrate host.

The Protozoa are generally divided into four main classes.

Class I.-SARCODINA

Protozoa in which the protoplasmic body has no limiting envelope in the form of a stiff cortical layer, thus tending to a spherical shape in the floating forms, or an irregular, ever-changing shape in the creeping forms. Organs serving for locomotion and capture of food are known as pseudopodia; a skeleton or shell may be present. Encysted resistant forms occur which may give rise to a number of daughter cells.

Sexual conjugation (gametogamy} is the usual process of reproduction, though autogamy within the cyst wall has been described.

EXAMPLES: Amœba limax, Entamœba coli. Entamœba histolytica.

Class II. MASTIGOPHORA

Protozoa in which the organs of locomotion and for food capture are flagella, that is to say, long slender filaments capable of performing lashing movements.

EXAMPLES: Trichomonas, Lamblia, Trypanosoma.

Class III.-SPOROZOA

Protozoa occurring as blood or intracellular parasites of other organisms. They possess no definite organs for locomotion or for ingestion of food. The typical reproduction takes place by the formation of spores and minute germs called sporozoites.

EXAMPLES: Gregarina, Coccidium, Plasmodium.

Class IV. INFUSORIA

Protozoa in which small filaments called cilia serve as organs of locomotion; these are distinguished from flagella by their much smaller size and in being present in great numbers, forming a fine covering over the whole of the body. The body protoplasm is always corticate.

EXAMPLE: Balantidium.

Class I. SARCODINA

This class, sometimes termed the Rhizopoda, includes parasitic amœbæ as well as free-living forms such as the Radiolaria and Foraminifera; these latter are of little interest to the student of tropical medicine. The distinctive characters of the amœbæ parasitic in man have already been given (p. 511). These are capable of independent amœboid locomotion by the alternate protrusion and retraction of protoplasmic processes called " pseudopodia."

Entamœba coli reproduces itself by binary fission in the ordinary way; this is called the vegetative or multiplicative phase. In addition to this a cystic or resting stage occurs, when conditions under which the parasite exists become adverse.

A sexual process, called autogamy, occurring within the cyst wall was described by Schaudinn, though recent authorities are inclined to doubt it. The process, as described, is as follows: The cyst contains at first but one nucleus, which divides into two. Each nucleus is then said to break up into chromidia and disappear, but to reappear later as a smaller and less distinct secondary nucleus on each side of the cell. Each secondary nucleus again divides into three; two of these again degenerate as reduction nuclei, while the third persists as a sexual or gameto-nucleus. The gameto-nuclei divide into four pronuclei, then two of each side conjugate and fuse, forming two nuclei or " synkarya" which subsequently divide into eight nuclei, in this manner forming eight amœbulæ within the cyst.

The end-product of the cystic stage of Entamœba histolytica possesses but four nuclei, and is known as the Entamœba tetragena of Viereck. The process of cyst formation by a budding process of the vegetative form, as described by Schaudinn, is now held to be incorrect.

The vegetative forms of the pathogenic and non -pathogenic species are further distinguished by differences in their protoplasmic contents. In E. histolytica the endoplasmic zone is granular, the ectoplasmic hyaline, while in E. coli no such differentiation occurs. The nuclei of the two forms have also distinctive differences. E. coli has a large and distinte nucleolus (karyosome) and a distinct nuclear membrane; whereas the E. histolytica nucleus is not so markedly differentiated (see p. 514).

Amœba limax and A. terricola are free-living forms of amœbæ occurring in water and earth. Their cystic stages possess a hard and refractile cyst wall. They are of importance to the tropical pathologist in that they may be ingested and recovered from the stools of dysenteries and others in culture on Musgrave and Clegg's medium. The parasitic amœbæ proper to man have not been so cultivated.

Class II. MASTIGOPHORA

The Mastigophora are divided into three subclasses, of which only one subclass, that of the Flagellata, containing the more typical forms, deserves our attention. The subclass Flagellata is divided into four orders:

i. Pantasomina.

ii. Protomonadina.

iii. Polymastigina.

iv. Euglenoidina.

The second and third only of these orders contain parasitic genera.

Order ii. Protomonadina

The Protomonadina are mostly flagellates of a small size with a principal and one or more subsidiary flagella. They are either saprophytic or parasitic.

Suborder 1.— Cercpmonas is a frequent parasite in the intestine of man; it is oval in shape, with a single nucleus and well-marked nucleolus, and has two flagella, one passing forwards, and the other backwards over the surface of the body to the hinder end, which is frequently drawn out into a protrusion resembling a tail.

Suborder 2.— Bodo (Fig. 225) is similar in shape and general structure to Cercomonas, but has two flagella, one directed forwards, the other backwards as a trailing flagellum; it occurs both as free-living forms and as parasitic in animals, for the most part in the digestive tract.

Suborder 3.— Prowazekia.— Prowazekia resembles Bodo in the arrangement of its flagella, but in the possession of a tropho- and a kinetonucleus it more resembles Trypanoplasma. It differs from Trypanoplasma in that the backward-directed flagellum is free from the body and devoid of an undulating membrane. Several species have been described from human fæces, and one, P. cruzi, is considered to be the cause of a diarrhœa in China.

Suborder 4.—The Hæmoflagellata and allied forms are an important suborder of the Protomonadina, and under this term are grouped a number of forms having a characteristic but not invariable parasitic habitat in the blood of vertebrates and in the digestive tract of invertebrates.

The group comprises a number of heterogeneous forms conveniently grouped together, their chief morphological feature being the possession of two nuclei, a tropho- and a kinetonucleus (blepharoplast). For this reason they were included by Hartmann as a distinct order of the Flagellata and termed the "binucleata."

The following five genera represent the more important types: (1) Trypanosoma, (2) Trypanoplasma, (3) Crithidia, (4) Herpetomonas, (5) Leishmania.

Fig. 225.—Bodo.
N., Nucleus; Rh., rhizoplast; Fl., flagella of unequal length.

1. The Genus Trypanosoma.—The structure of the trypanosome body is of a uniform type, though subject to variation in minor details. The body is long and sinuous; the anterior^[2] end tapers gradually to a narrow point, while the posterior end generally terminates more bluntly.

The principal nucleus (trophonucleus) is situated centrally; the kinetonucleus is usually placed posterior to the nucleus, but is sometimes closely approximated to it. Certain exceptions to this rule are known, namely, the recently described T. rhodesiense (p. 185), and some multiplicative forms of T. lewisi. The flagellum arises from a centriole (blepharoplast) closely connected with the kinetonucleus, and passes forwards as the margin of the undulating membrane; in some cases it may end with the undulating membrane at the anterior extremity of the body, but more usually it projects forwards as a free lash. The arrangement of the centriole and kinetonucleus varies; sometimes the former is lodged within the latter, sometimes connected with it by a delicate rhizoplast. In one species, T. equinum (Fig. 226), the cause of "mal de caderas" in South America, the kinetonucleus is very minute.

Fig. 226.
T. equinum.
(After Laveran.)

Trypanosomes occur as blood parasites in all classes of vertebrates. Many wild animals harbour small numbers of these parasites in their blood. Certain are specific and probably harmless to their particular vertebrate host.

Trypanosomes of cold-blooded animals include T. rajæ, T. damoniæ, T. granulosum, and T. rotatorium (Figs. 227-30).

Though showing but slight morphological differences as a general rule, members of this genus have been differentiated on morphological as well as on pathological grounds.

Fig. 227.—T. rajæ. (After Laveran and Mesnil.) A trypanosome of fish (the skate).	Fig. 228.—T. damoniæ. (After Laveran and Mesnil.) A trypanosome of the tortoise (Damonia revesi).	Fig. 229.—T. granulosum. A ttrypanosome of the eel.
Fig. 230.—T. rotatorium. (After Laveran.) A trypanosome of the frog (Rana esculenta).

The Lewisi group includes T. lewisi, T. cuniculi, T. duttoni of the mouse, T. microti of Microtus arvalis, all about equal in size (24-25 μ in length and 1-2 μ in breadth), and similar in general appearances; that is, the organisms are long and slender; both the flagellar and aflagellar extremities are markedly pointed and the nucleus situated towards the flagellar end. They are non-pathogenic, or only slightly pathogenic.

The Brucei group includes nearly all the pathogenic varieties: T. brucei (nagana), T. gambiense (sleeping sickness), T. rhodesiense^[3] (human trypanosomiasis in Rhodesia), T. evansi (surra), T. hippicum (murrina, a disease of mules in Panama), T. equinum (mal de caderas), T. equiperdum (dourine, or mal du coït), T. vivax vel cazalboui (souma disease of mules and cattle in the Soudan).

These trypanosomes are all about the same size (22-28 μ in length and 1-3 μ in breadth). The aflagellar extremity is blunted to a variable extent, the nucleus is centrally placed, and the protoplasm granular. For further details of the members of this group, see p. 194.

According to some authors, T. dimorphon, a parasite of horses in the Gambia, producing symptoms resembling nagana, represents a distinct type. As its specific name implies, it is found in two distinct forms—one long form, 22 μ in length; and a short, stumpy form, 1-5 μ in length. The aflagellar extremity is markedly blunted, and the body protoplasm is continued to the end of the flagellum.

T. nanum resembles T. dimorphon in general features, and is the smallest of all the mammalian trypanosomes, measuring 11-16 μ by 2·5 μ. It produces a chronic disease, marked by emaciation and anæmia, in cattle in the Soudan.

T. theileri (Fig. 231), 60-70 μ in length by 4 μ in breadth,

Fig. 231. T. theileri. (After Laveran.)	Fig. 232. T. lewisi. (After Laveran.)	Fig. 233. T. brucei. (After Laveran.)

one of the largest mammalian trypanosomes, is recognized by its large size and rapid movements. It is apparently non-pathogenic, and is transmitted, according to Theiler, by Hippobosca rufipes. T. ingens, found in cattle in Uganda, is still larger than T. theileri, measuring 72-122 μ in length, and the flagellum alone measuring 17 μ.

The transmission of these trypanosomes from one host to another is carried out, so far as is known, by the agency of some blood-sucking invertebrate. When such a host is of terrestrial habit the definitive host is an insect, when aquatic a leech; though in one species, T. equiperdum, the cause of "dourine" in horses, the trypanosomes may pass from one host to another during coitus. Hereditary transmission in trypanosomes has not yet been proved. One or more species of insect may serve as the definitive host; thus in T. lewisi the selective hosts are the rat-fleas (Ceratophyllus fasciatus and Xenopsylla cheopis), though it may also develop in a different genus, the rat-louse (Hæmatopinus spinulosus), and also in Pulex irritans and Ctenocephalus canis.

The life cycle in the vertebrate host is generally as follows: After infection (as in T. lewisi, Fig. 232) the trypanosomes appear in the blood about the fifth to the seventh day. Owing to the rapidity with which fission takes place, the forms are of great variety. After two or three months have elapsed the swarms of trypanosomes in the rat's blood have begun to diminish and eventually disappear. When this has taken place that rat is immune to that particular species of trypanosome.

No confirmation of Schaudinn's statement that some part of the development of trypanosomes in the blood is ultra-microscopic has been made, though nitrations of infected blood plasma through a bacterial filter have been experimented with. Fry and Ranken have observed a process of granule-shedding in trypanosomes under the dark-ground illumination (see p. 161).

In some instances trypanosomes appear in the peripheral circulation only at certain periods, as in the case of T. rhodesiense in man; and in others, as in the trypanosome of Athene noctxa, the little owl, they are said to be most numerous in the peripheral circulation during the night-time, and then only during the summer months; in the winter months they are found in the bone marrow.

The cycle in the invertebrate host takes place mainly in the digestive tract. Thus, T. lewisi, after being imbibed by the flea, multiplies rapidly, chiefly in the stomach cells, subsequently passing into the hindgut and rectum. Here the parasites assume a leptomonas and crithidia appearance, and occur in large bunches attached by their flagellar ends to the epithelium, where they divide with great rapidity, or they may occur in cyst-like masses within degenerating epithelial cells. Eventually they give rise to the small infective trypanosomes first described by Swellengrebel and Strickland, which occur in vast numbers in the hind-gut and rectum of the flea. The latter becomes infective in four to seven days, and it may remain infective for forty -five days or longer. The flea does not infect the rat through its proboscis, and the parasites are not found in the flea's salivary glands. Theoretically infection can take place in three ways: (a) the flea harbouring the infective forms of the flagellates may be crushed and devoured by the rat; (b) the rat may lick its fur upon which an infected flea has just dejected; (c) the rat may lick and infect with flea dejecta the wound produced by the insect. Of these the second is probably the most common method; the other two are doubtful.

Many pathogenic trypanosomes in Africa are transmitted by tse-tse flies (Glossina), and probably these flies remain infective for life. Thus, T. gambiense of man is transmitted by Glossina palpalis, and perhaps by G. morsitans as well; T. brucei (T. pecaudi) (Fig. 233) of big game, and T. rhodesiense of man, by G. morsitans; T. vivax (probably identical with T. cazalboui) by G. morsitans, palpalis, longipalpis, and tachinoides; T. caprœ by G. morsitans; T. dimorphonby G. palpalis; T. grayi, the trypanosome of crocodiles, is also transmitted by this same glossina; T. evansi, of "surra," a disease of horses in India, by Stomoxys nigra, and probably by a tabanus as well. The trypanosomes of birds probably develop in mosquitoes; those of fishes and turtles in leeches.

Two methods of transmission of trypanosomes must be distinguished. The one, a mechanical method, can be performed by any blood-sucking insect for a few hours after a meal of infected blood; the other, indirect or cyclical, is a developmental cycle, and can only take place in the particular definitive host—that is, in most instances, some species of glossina—to which the particular species of trypanosome has become adapted.

Fig. 234.
T. gambiense

The development of T. gambiense (Fig. 234) in G. palpalis, thanks to the researches of Kleine, Taute, Bruce and his collaborators, is now fairly completely known. Apparently the whole development takes eighteen to twenty-five days or more. Five to seven days after ingestion of infected blood the trypanosomes become scarce in the digestive tract of the fly, only to reappear in large numbers and in a variety of crithidial and other forms. Finally an invasion of the salivary glands by short, stumpy, trypaniform individuals takes place. In T. vivax (cazalboui) a completely different mode of development in glossina takes place, a method termed by Roubaud évolution par fixation directe; this occurs solely in the proboscis of Glossina palpalis. The trypanosomes assume a leptomonas shape and attach themselves by their flagellum to the labium or hypopharynx of the proboscis tube, multiplying in the salivary fluid; these flies remain infective for life. A third method of development occurs in the case of T. dimorphon—the évolution par fixation indirecte of Roubaud. In this case the trypanosomes multiply first in the digestive tract of the glossina, and then pass forwards into the proboscis.

In T. cruzi the development both in the intermediary and in the definitive host is of so different a character that Chagas has relegated it to a special genus, Schizotrypanum. In man the adult trypanosomes multiply not in the peripheral circulation but in the internal organs of the body. In the capillaries of the lungs the adult trypanosome loses its flagellum and occasionally the kinetonucleus as well, the body becomes round, and the mass breaks up into eight small individuals (merozoites). Some regard these merozoites as exhibiting a sexual differentiation. The merozoites enter the red blood-cells, and so gain the general circulation. Within the corpuscles they form into a normal trypanosome, which, when set free, either repeats the schizogonic cycle or enters into the invertebrate host, in which it develops still further.

A second type, and probably the normal method of multiplication in man, takes place within the hypertrophied endothelial cells of the lung and striped muscles, notably the cardiac muscle, but can apparently occur in any tissue. In this situation the parasite assumes the shape and appearance of a leishmania.

The developmental cycle of this trypanosome takes place in a bug, Lamus megistus, and is shortly as follows: The trypanosomes taken up into the bug's stomach change in about six hours; having lost their flagellum they assume a leishmania form and multiply by fission. After a while these forms assume a crithidial shape and pass into the intestine, eventually gaining the body cavity and salivary glands of the insect as small trypanosomes, and in this manner pass into the vertebrate host.

Fig. 235.
Trypanoplasma borreli.
(After Laveran and Mesnil.)

The changes undergone by trypanosomes in culture on N.N.N. medium (p. 158) are diverse and striking; they then exhibit a series of forms quite distinct from those seen in the blood of the vertebrate, but bearing more resemblance to those seen in the invertebrate host. In such cultures they assume crithidial, leptomonas, and even leishmania forms.

2. The Genus Trypanoplasma (Fig. 235).—The species of this genus parasitic in blood are only known as yet to occur in fresh-water fishes, and are transmitted by leeches. A number of species are parasitic in the organs of snails, such as T. helicis, found in the receptaculum seminis of Helix pomatia. The principal feature is the presence of two flagella arising together from two blepharoplasts at the anterior extremity. One flagellum projects forwards, passing down the side of the body to the hind end, forming the free edge of the undulating membrane and projecting freely backwards as a free flagellum.

Fig. 236.—Crithidia.
Fl., Flagella; M., undulating membrane; Bl., blepharoplast; N., nucleus.

3. The Genus Chithidia (Fig. 236).—The distinctive feature of this genus is the relatively short undulating membrane and flagellum arising from the centre of the body at a point in close proximity to the kinetonucleus. The shape of the body varies considerably. Crithidial stages are commonly found as developmental forms of trypanosomes. There remain, however, a number of forms which are parasitic in the intestinal tract of insects, notably the Chironomidæ and Pulicidæ.

4. The genus Herpetomonas (Leptomonas).—Intestinal parasites of insects, especially muscidæ and hemiptera. In most cases they are parasites of insects alone, infection being brought about by the ingestion of cyst-infected fæces. Some species are parasitic in the latex of plants, and are taken up by the appropriate plant hemiptera. The distinctive characters (Fig. 237) are the possession of a single flagellum arising from a single kinetonucleus, itself situated in the anterior portion of the body, and the absence of an undulating membrane. Uniflagellate and biflagellate forms occur. Some authorities regard the latter as the result of precocious formation of a new flagellum in anticipation of longitudinal

Fig. 237.—Herpetomonas muscæ domesticæ. (After Prowazek.)
Fl., Flagella; Bl., blepharoplast; N., nucleus; Ax., axostyle.

division. They have shown that trypanosome forms, but lacking the well-developed undulating membrane so characteristic of the mammalian trypanosome, also occur, the changes in form being in all probability brought about by the migration of the nuclei; all stages being found between the non-flagellate leishmania, through leptomonad, crithidial, to the trypanosome form (Fig. 240).

Fig. 238.—Leishmania donovani. (After Leishman.)

5. The genus Leishmania includes human parasites, L. donovani (Fig. 238), the cause of a general disease, kalaazar, and L. tropica, the cause of oriental sore—a purely local infection. L. donovani, in the Mediterranean area, is found especially in children, and is associated with a similar disease occurring in dogs and caused by the same parasite. The type of parasite found in the vertebrate host is a small oval body multiplying by fission and contained within cells—endothelial cells or macrophages; it is very uniform in size. Each parasite possesses a tropho- and a very distinct kinetonucleus (blepharoplast).

In cultures on the N.N.N. medium the leishmania increase in size, and develop a flagellum, finally assuming an elongated leptomonas form. It is thought that this latter represents a stage of the development of the parasite in the invertebrate host; such a development, however, has not yet been followed. Patton believes that in the case of L. donovani the parasite completes its developmental cycle in Cimex rotundatus, and in the Mediterranean area there is a certain amount of evidence to incriminate Ceratophyllus fasciatus, the dog-flea, as being the definitive host. In the case of L. tropica, it has been pointed out that since the sores, as they occur in man, are situated in some uncovered part of the body, the invertebrate host is more likely to be some biting fly or mosquito, or even the sand-fly (Phlebotomus).

Morphologically the leishmania is a leptomonas, but the fact that it occurs in a vertebrate host should justify its inclusion in a separate genus, Leishmania.

Fig. 239.—Diagram of types of flagellates. (After Wenyon.)
a, Trypanosome; b, Crithidia; c, Leptomonas; d, Leishmania.

Nomenclature of the hæmoflagellates.—The generic terms given to these different parasites are also utilized to denote the different stages in their life cycles. Of this cycle the leishmania is taken to represent the most primitive, the trypanosome of vertebrates the most highly developed form (Fig. 239).

One and all these stages occur in the developmental forms of certain trypanosomes in culture, and in the gut of the definitive host the glossina, and even in the vertebrate, as in T. lewisi. The generic titles Trypanosoma, Herpetomonas, Crithidia and Leptomonas should, strictly speaking, be reserved for the parasite according to the highest developmental form it is capable of assuming. The accompanying diagram, taken from Wenyon (Fig. 240), illustrates this idea.

Parasites of Uncertain Zoological Position resembling Leishmania

Hæmocystozoon braziliense was found by Franchini in the blood of a man from Brazil. The parasites are sometimes oval, the size of a red blood-cell, containing a large nucleus, a variable amount of pigment, and one

Fig. 240.—Diagram of the classification of the trypanosomes and allied flagellates. (After Wenyon.)

or more centrosomes, and sometimes a terminal flagellum arising from a blepharoplast; sometimes non-flagellate and very minute forms were found in the red cells. In a liver puncture, round, clear and encysted forms occurred, enclosed exceptionally in lencocytes. The patient suffered from irregular fever and anæmia combined with a cystic enlargement in close proximity to the inferior cervical glands. Liver and spleen were also enlarged.

Histoplasma capsulatum was found by Darling in Panama in the tissues of one Chinaman and two negroes who died of some obscure malady characterized by wasting, irregular fever, enlarged spleen, and anæmia. In smears and sections of the lung small bodies were found within the epithelioid cells which bore a great resemblance to leishmania, but differed in possessing no kinetonucleus (blepharoplast). According to Rocha-Lima the parasite is really a yeast-like organism and has nothing to do with the protozoa, but resembles the Cryptococcus farciminosus, the cause of epizootic lymphangitis of horses in Senegal.

Order iii.—Polymastigina

Polymastigina possess three or more unequal flagella; a distinct mouth opening may be present or absent; in other points they resemble the foregoing. The Polymastigina are divided into two divisions or suborders—the Tetramitidæ and the Octomitidæ.

Fig. 241.—Trichomonas intestinalis. (After Martin and Robertson.)	Fig. 242.—Lamblia intestinalis. (After Wenyon.)
Bl., Blepharoplast; Ch. L., chromatinic line; M., mouth; Fl., ant. flagella; N., nucleus; Ch. B., chromatic body; Ax., axostyle; U. M., undulating membrane.	N., Nuclei; Ax., axostyles; Fl. 1-4, four pairs of flagella.

Suborder 1.—The Tetramitidæ have three or more flagella arising at the anterior end; the trailing flagellum, if present, may be united to the body by an undulating membrane.

Trichomonas intestinalis (Fig. 241), a common parasite in man, has the trailing flagellum united to the body by such an undulating membrane. It is a matter of controversy whether encystment occurs. Trichomonas, in addition to its motile powers, is capable of a certain amount of amœboid movement; it possesses a definite but small mouth cavity, and a stiff supporting rod known as the axostyle.

Tetramitus mesnili also occurs as a harmless parasite in human fæces; it resembles trichomonas in its general features, but differs in the lack of an undulating membrane and in the presence of a well-marked cystosome in which there is either a flagellum or a membrane; there are three anterior flagella as in tnchomonas.

Suborder 2.—The Octomitidæ, for the most part intestinal parasites, have six or eight flagella, generally arranged in pairs; the body is bilaterally symmetrical in structure.

Lamblia intestinalis (Fig. 242) is a common parasite in the human intestine; encystment is a common feature; these cysts are ovoid bodies containing two nuclei. On the ventral surface of the free form there is a sucker-like depression. The general structure of the parasite is sufficiently well shown in Fig. 242.

Class III. SPOROZOA

This class contains four orders of interest:

i. Gregarinidea.

ii. Eimeriinea.

iii. Hæmosporidia.

iv. Neosporidia.

Order i.— Gregarinidea

This order contains the family of Gregarines. The young parasite (trophozoite) is at first a parasite of the endothelial cells, but later, becoming free, lies in the body cavity and in the intestinal canal. The full-grown parasite is of a large size, and has a definite shape and cuticle. Gregarines are found commonly parasitic in the digestive tract of insects; they are not known to occur in the vertebrata. The anterior end of the body may be provided with a rostrum, termed the epimerite, armed with hooks and processes; the posterior portion of the body may be divided by a single septum into two segments, known as the protomerite and the deutomerite; the nucleus, often of a large size and oval shape, is situated in the latter portion. The complete parasite is capable of independent rapid motion.

The Gregarines are divided into two groups, on one of which, Eugregarinæ, a sexual phase— sporogony— only takes place. In the other, Schizogregarines, multiplication is effected by both sporogony and schizogony.

The Eugregarines are further divided into two groups, the cephaline and acephaline gregarines. The cephalines have a well-developed epimerite; in the acephalines the body is not segmented.

In the sporogonic phase two full-grown gregarines approximate to one another, become rounded, and proceed to secrete a tough envelope. Each gregarine breaks up into a number of gametes which conjugate in pairs. The resulting zygotes (sporoblasts) each encyst in a sporocyst in which eight sporozoites are formed; these on entering a new host develop

into adult gregarines. The whole cycle takes place within the same insect or earthworm.

Order ii.— Eimeriinea

This order also consists of intracellular parasites. A number of spores are produced within a cyst, itself the development of a single zygote. There is an alteration of generations, non-sexual schizogony alternating with a sexual cycle or propagative schizogony.

The Eimeriinea are divided into a number of suborders, amongst which are the Eimeriidea (or true Coccidia), the Hæmogregarinidæ, and the Adeleidæ.

Suborder Eimeriidea.— The typical life-cycle of a coccidium is that of C. schubergi in the centipede Lithobius forficatus. The young parasites or sporozoites are liberated from a cyst in the intestinal tract and penetrate epithelial cells, where they grow into large schizonts, characterized by a large vesicular nucleus and a karyosome (nucleolus) . When full grown the nucleus divides by repeated fission till a variable number, generally about thirty, are produced. The schizont now divides into as many merozoites as there are nuclei, and is then full grown. The cells burst, the merozoites are set free, and, entering other cells, may develop in two ways, either into schizonts again or into sporonts. The sexes of the sporonts can be distinguished; in the male the protoplasm is clear, but in the female it is crowded with reserve food material.

The male sporont develops further; the nucleus gives off a number of chromidial bodies which become aggregated together to form secondary nuclei; the nuclei develop into microgametes— small, slender bodies provided with two flagella. The host cell then bursts, liberating the microgametes, which endeavour to enter the female cell or macrogamete. When one such has effected its entrance the macrogamete secretes a tough membrane and becomes an oöcyst, thus effectually preventing the entrance of any other microgametes. The penetrating microgamete fuses with the female nucleus, forming a synkaryon; fertilization is then complete, and in this condition the oöcyst is passed from the original host and falls to the ground, where further development takes place. The zygote breaks up into a number of sporoblasts; each sporoblast secretes a tough envelope, the sporocyst. Therefore, when sporogony is complete, the original oöcyst contains four sporocysts, each containing two sporozoites. In order to develop further the oöcyst must be swallowed by a new host, whereupon the tough membranes dissolve, liberating the sporozoites.

Coccidia are common parasites of vertebrates (rabbits and fowls), but are also found in invertebrates.

Suborder Hæmogregarinidæ.— Although, strictly speaking, parasites of the blood of vertebrates, these parasites have been relegated, apparently on adequate grounds, to this suborder. Their life cycle in many particulars resembles that of the Adeleidæ in that the male and female gametocytes are associated, but differs in that the sexual cycle is passed in an invertebrate host instead of in an oöcyst on the ground. The Hæmogregarines are found in all classes of vertebrates, and are especially common in reptiles, amphibia, and fishes, but are also found in certain mammals, such as dogs, cats, rats, and the jerboa. They are sausage-shaped bodies with a large central nucleus, and lie encapsuled in the red blood-cells or even in the leucocytes (H. canis and H. muris). They are not

Fig. 243.—H. seligmanni: encysted sporont, showing operculate capsule. (After Sambon.)	Fig. 244.—H. seligmanni: free sporont. (After Sambon.)

amœboid and no melanin is produced. The sporont, which is the form seen in the peripheral blood, is liberated from the corpuscle as a free vermicule, much resembling a small gregarine in appearance and in its gliding movements. Schizogony does not proceed in the blood, but, as in the case of the subtertian malaria parasite, in some internal organ—in

Fig. 245.—H. jaculi. (After Balfour.)
a, Normal erythrocyte of jerboa; b, sporont; c-h, schizogony within liver cells.

the bone marrow in some animals; but in the case of H. jaculi of the jerboa, and H. muris of the rat, in the liver cells (Fig. 245.) A number of merozoites are produced; this number varies. Two distinct types of merozoites are formed within different cells. In one type they are numerous and slender, and are then termed micromerozoites; in the other they are few in number and stout, and are then called macromerozoites. The former, re-entering red cells, are destined to become sporonts; the macromerozoites may again repeat the cycle and become once more macromerozoites or micromerozoites.

These sporonts become differentiated into male and female gametocytes and appear in the circulating blood, but only develop further in the invertebrate host. In the intestine of the invertebrate the male and female gametes, having escaped from their cells as motile vermicules, become approximated and fuse to form a zygote (but in the case of H. stepanowi of the tortoise four microgametes are first produced by the male gamete). The zygote then apparently becomes an oökinete, encysts, and grows into a large oöcyst containing a variable number of sporocysts, each of which contains six or eight sporozoites, which again in some manner pass into the intermediary host. In the case of the leech the exact manner in which this is performed is not known, but in the case of H. muris, which develops in the rat-mite (Lelaps echidninus), the mite is devoured by the rat, and the sporozoites, liberated in the rat's intestine, pass through the wall of the gut into the blood-stream, re-enter the liver cells, and become schizonts.

Fig. 240.—H. canis; endocellular in leucocytes and free sporonts.
(After Wenyon.)

In the leucpcytic gregarines, of which the beat known is H. canis(Fig. 246), described by Christophers and Wenyon, schizogony takes place in the bone marrow and spleen of the dog, and is, as described above, of two distinct types. The macromerozoites in this case pass into the blood, are taken up by the leucocytes, and become gametocytes. Sporogony takes place in the body tissues of the tick Rhipicephalus sanguineus. The dog probably becomes infected by eating the tick.

Suborder Adeleidæ.—This suborder is of little interest from the standpoint of human pathology. It consists of intracellular parasites of invertebrates which differ in their life-history from the foregoing in that the sporonts do not remain separate as in Coccidium schubergi, but associate in pairs, the male sporont becoming attached to the female.

Order iii.—Hæmosporidia

These parasites are parasitic during the greater part of their life cycle; but they exhibit, as do the hæmogregarines, an alternation of generation, sporogony generally taking place in the digestive tract of some invertebrate.

The order comprises the genera Plasmodium, Hœmoproteus, Leucocytozoon, and Piroplasma.

1. In the genus Plasmodium the young schizonts occur within red blood-cells, exhibit amœboid movement, and

Fig. 247.—Plasmodium vivax: schema of entire life-cycle.

produce a pigment called "melanin." The invertebrate host is generally a mosquito. Such are the parasites of human malaria—the benign tertian Plasmodium vivax (Fig. 247), the benign quartan Plasmodium malariæ, and the pernicious or subtertian Plasmodium falciparum (formerly called Laverania malariæ) (Fig. 248). All these human parasites undergo sporogony in mosquitoes of the genus Anopheles. Similar parasites are found in monkeys (Fig. 249), antelopes, bats, and squirrels. In birds an analogous and pernicious parasite is known as Proteosoma (Proteosoma grassii, Labbé, 1894), but, in contradistinction to the human malaria parasite, it is transmitted by mosquitoes of the genus Culicinæ, and also by Stegomyia calopus.

The life cycle of these parasites (Fig. 247) is extremely well known and has already been described in detail (p. 28).

Fig. 248.—Plasmodium falciparum.

The sporozoites, minute slender organisms, are introduced by the proboscis of the mosquito, and enter red blood-corpuscles. The young trophozoite is characterized by its signet-ring appearance, there being a large space in the centre of the body. As the parasite grows this space disappears, and the pigment is deposited in its protoplasm. When full grown, multiplication by schizogony takes place. The nucleus, hitherto single, multiplies by repeated division, each daughter nucleus becoming the centre of a merozoite. A blood corpuscle containing a number of such merozoites represents the characteristic rosette of the malaria parasite. The host cell then

Fig. 249.—Plasmodium kochi. (After Lühe.) From a monkey
(Cercopithecus sabacus).

ruptures, setting free the merozoites; these penetrate other corpuscles and become trophozoites, which may either grow into schizonts or sporonts. These sporonts (gametocytes) are either male or female: the male forms have a large nucleus and a hyaline faintly-staining protoplasm; the female forms have a smaller nucleus and a deeply staining protoplasm. The sporonts are capable of further development only if taken up by the specific kind of mosquito. In the male the nucleus undergoes disintegration, and at the same time the fragments proceed to the periphery of the cell and become nuclei of a number of fine filaments or flagella, and two polar bodies are simultaneously extruded; these flagella are endowed with motile powers, and break free from the cell as microgametes. In the meantime the female sporont, after giving off a reduction nucleus, is ready for fertilization by the microgamete. The female cell (zygote) is capable of independent movement, and (now termed an oökinete) bores its way through the lining epithelium of the mosquito's stomach, there encysts between the epithelium and the limiting membrane, and becomes an oöcyst. The original nucleus now breaks up into sporoblasts, which in turn break up into sporozoites. The oöcyst then bursts, setting free the sporozoites, which contrive to pass into the salivary glands, whence, with the salivary secretion, they once more enter the blood on which the infected mosquito is feeding. In the avian red blood-corpuscle the young sporont of proteosoma displaces the nucleus of the cell to one side in its growth, and thus may be distinguished from a similar stage in Halteridium.

A form of pigment-producing plasmodium occurs in the blood of reptiles, and is known as Hæmocystidium (Fig. 250). These parasites are of large size, are very numerous in the blood, and do not exflagellate when the blood is drawn. Nothing further is known of their life-history.

Fig. 250.—Hæmocystidium simondi.

Toxoplasma, a parasite of small size inhabiting the endothelial cells of the peritoneum of mono- and polymorphonuclear leucocytes, was discovered by Splendore in an epizootic amongst rabbits in Brazil. It has since been found in pigeons and in a rodent called the gondi (Ctenodactylus gondii) by Nicolle in Tunisia. Multiplication takes place by binary fission; the nucleus is single; no blepharoplast is present. From its morphology it is apparently allied to the hæmamœbæ, but has been relegated provisionally by Nuttall to the piroplasmata.

2. Members of the genus Hæmoproteus (also termed Halteridium) are parasitic in the blood of many widely different species of birds, and perhaps also of turtles. Within the red blood-corpuscle they grow into a characteristic halter-like shape, partially enveloping but not displacing the nucleus of the corpuscle. In the halteridium of the pigeon the sexual cycle is passed in a hippoboscid fly, Lynchia maura, and is similar to that described above, but differs in one respect, namely, that the melanin pigment is extruded by the oökinete, which becomes considerably larger than the sporont found in the blood of the bird, and in this stage is apparently re-inoculated by the lynchia into the pigeon. Possibly the further development of the oökinete takes place within a capillary endothelial cell of the pigeon, but it is afterwards found within a leucocyte in the lung capillaries. In this leucocyte the parasite grows and breaks up into a number of small forms or merozoites; finally the leucocyte bursts, setting free the merozoites, which, entering red blood-corpuscles, assume once more the typical shape of the sporonts. The male sporont is easily distinguished from the female by the larger size of its nucleus and by the faintly staining properties of its protoplasm.

The life-cycle as worked out by Schaudinn was of an entirely different character. It is now known, that the blood of Athene noctua, the little owl, with which Schaudinn worked, contains a number of blood parasites, namely, a proteosome, a halteridium, two trypanosomes, a lencocytozoon, and a spirochæte. Schaudinn held that the trypanosomes, the leucocytozoon, and the spirochæte formed different stages of the same life-cycle.

3. The genus Leucocytozoon of Daneliewsky must be carefully distinguished from the leucocytic hæmogregarines to which allusion has already been made. The leucocytozoa are elongated oval bodies (Fig. 251), parasitic in the blood of birds. They modify the shape of the host cell, generally a mononuclear leucocyte. Male and female forms are recognized; in the latter the protoplasm takes on a deep stain; no

Fig. 251.—Leucocytozoon neavei. (After Neave.) From the guinea-fowl.

pigment is formed, Schizogony takes place in the spleen of the bird. Exflagellation and fertilization of the gametocytes occur exactly as in the malaria parasite, but their method of transmission and the definitive host are still unknown.

4. The genus Piroplasma consists of a number of minute oval or rod-like organisms, several of which are often contained in the one red blood-corpuscle. No pigment is produced, but the corpuscle is destroyed, the hæmoglobin set free and ultimately excreted by the kidneys of the host.

The best-known piroplasm is Babesia bovis (or bigemina), the parasite of red-water fever of cattle.

Piroplasma caballi causes biliary fever in horses; Pircplasma (Babesia) bovis, red-water or Texas fever in cattle; Piroplasma canis (Fig. 252), malignant jaundice in dogs; while Piroplasma mutans is apparently a harmless parasite in the blood of African cattle.

The typical piroplasma is a pear-shaped body dividing by a process of budding, and is found in the red corpuscles of oxen, of sheep, and of a number of wild animals.

The genus Piroplasma has, for convenience' sake, been divided into a number of subgenera of doubtful validity.

Theileria is the title reserved for a number of bacillary or coccoid forms. Theileria parva (Fig. 253) is the parasite of "East Coast fever" of African cattle.

Another piroplasm, P. mutans, is commonly found in the blood of cattle suffering from "East Coast fever," together with the specific parasite Th. parva. In the case of the latter the forms in the blood consist entirely of gametocytes; in P. mutans, on the other hand, the quadruple forms in the

Fig. 252.—Piroplasma canis. (After Nuttall.)

erythrocytes apparently represent a stage of the schizogony. Therefore direct inoculation of blood containing Th. parva does not produce the disease, whereas infection with P. mutans invariably occurs, if schizonts of this species are present in the inoculated blood. The schizogonic stage of Th. parva is apparently passed in the internal organs such as the spleen. Splenic puncture in the diseased animals reveals bodies bearing a rough resemblance to a sporulating quartan malaria parasite known as "Koch's bodies"; it is thought that these bodies, which are capable of reproducing the disease on re-inoculation, represent the schizogonic cycle in the splenic cells.

Fig. 253.—Theileria parva. (After Theiler.)

Nuttallia is a small oval or pear-shaped piroplasm, also multiplying in the form of a cross. Two kinds are known: one, N. equi, causes piroplasmosis in horses in southern Europe; the other, N. herpestidis, is found in the mongoose.

Anaplasma, a small coccoid body, perhaps to be included with the piroplasms and apparently causing great destruction of the red cells, has been described by Theiler in cattle. These bodies are arranged either in the centre of the cell or radially; hence the names A. centrale and A. marginale have been applied to them. According to some authorities they are not parasites at all, but merely represent a degeneration of the red cell.

Within the red cell the piroplasm consists of little save vacuolated protoplasm and a simple nucleus. The parasites are said to exhibit a certain amount of amœboid movement, and, when free from the cell, to be capable of independent locomotion. The development in the vertebrate host appears to proceed solely by either binary or quadruple fission.

The transmission of the piroplasms is effected by ticks; in the case of P. bovis it is adapted to the life-history of the transmitting tick (Ixodes reduvius or Magaropus australis), and in P. canis to Rhicephalus sanguineus. The tick is hatched first as a minute six-legged larva, which becomes an eight-legged nymph, differing from the adult only in the absence of genital organs. In each stage the tick feeds but once. The infection is passed to the ova by the infected female tick. The developmental stages of this parasite in the tick are little known; apparently, according to Christophers and Koch, the parasites escape from their red cells and become transformed into starshaped bodies representing the gametocytes. Conjugation of these bodies takes place, and when complete the zygote becomes a motile body resembling an oökinete. The oökinetes apparently penetrate into the ova of the tick, when they

Fig. 254.—Developmental forms of Piroplasma canis in tick's gut. (After Koch, Klein, and Christophers.)

assume a globular shape. When the egg hatches into a six-legged larva these bodies break up into a number of sporoblasts, which become scattered throughout its tissues. The sporoblasts divide in turn into a number of sporozoites resembling the pear-shaped forms seen in the blood; these, according to Christophers and Gonder, contrive to enter the salivary glands, and thus re-enter the blood-stream when the tick becomes adult and again feeds on blood. (Fig. 254.)

Order iv.— Neosporidia

Parasites belonging to this order are developed from small amœbulæ within the body of the one host. No definitive host is known. The adult parasite is a large cyst contained within a definite membrane and incapable of independent movement. The motile amœbula is followed by a plasmodial intracellular stage in which spore formation is commenced and is continued throughout the life of the parasite. The order is divided into two suborders: (1) the Cnidosporidia, characterized by the possession of polar capsules, and pear-shaped bodies provided with a spiral and delicate filament; (2) the Haptosporidia, in which those features are absent.

The life -history of Sarcocystis tenella, a Cnidosporidian, will be considered. It is a parasite of vertebrates, sometimes occurring as a cystic white body, known as "Miescher's tubes," visible to the naked eye in the striped muscles of man. As a rule it produces no symptoms. The cyst contains a number of crescentic bodies (spores); in some cases these spores are motile; each spore is provided with a nucleus situated at the blunt posterior end and a striated polar capsule at the anterior end. In this species the mode of propagation from one host to another is unknown, but mice can be infected by feeding on the bodies of others harbouring S. muris; apparently in this case the spore germinates in the intestine of its host and sets free an amœbula which eventually reaches the striped muscle. In this situation the nuclei break up to form a plasmodium, and become the central points of many separate cells, called pansporoblasts or sporonts. These sporonts multiply actively and occupy the peripheral zone of the parasite, whereas the central portion is packed with spores in the process of differentiation.

Of the Haptosporidia, one, Rhinosporidium kinealyi, is found in man in India. It gives rise to polypoid outgrowths from the septum nasi and from the external auditory meatus. The youngest parasites appear to be round cells provided with a cuticle and a single nucleus. By repeated division of this nucleus the parasite becomes a multinucleate plasmodium. The cyst contents become divided into pansporoblasts in the centre, while the periphery contains the granular nuclei. The sporonts grow in size, and by repeated division form clusters of spores, a kind of a morula enclosed in a membrane. When ripe the cyst bursts and scatters its contents into the surrounding tissues.

Nothing is known of the transmission of this parasite or the manner in which infection is acquired.

Class IV.—INFUSORIA

The Infusoria are represented by Balantidium coli, an oval-shaped parasite belonging to the subclass Ciliata. The parasite measures about 60-100 μ in length by 50 μ in breadth. The body is clothed with a thick covering of cilia. There is a lobulated nucleus with a distinct micronucleus lying close to it. The protoplasm contains a variable number of contractile vacuoles; there is a definite anteriorly-situated cystosome or mouth cavity. Nutrition is effected by ingestion of solid particles and by osmosis. The parasite reproduces itself asexually by transverse fission. Conjugation takes place by approximation of two individuals and by the exchange of certain nuclear elements; after this has been effected the conjugants separate. Encystment may take place.

Balantidium coli burrows into the submucosa and causes dysenteric symptoms known as balantidial dysentery (see p. 523).

DOUBTFUL GROUPS

Two groups of protozoal organisms of doubtful validity remain to be considered, the Spirochætes and the Chlamydozoa.

1. The Spirochætes

Regarding the systemic position of the Spirochætes a great deal of controversy has been waged during recent years (see p. 232). The group comprises a number of types, some of which are parasitic, others free-living forms, such as Spirochœta plicatilis and Cristispira balbiani of the oyster. Both possess a crest running the entire length of the body; this structure has been wrongly termed an " undulating membrane."

The pathogenic spirochætes are slender thread-like organisms containing chromatic material. Such are Sp. recurrentis (obermeieri), S. duttoni of relapsing fever, Sp. gallinarum of fowls, etc.; these species are transmitted by various kinds of ticks (Ornithodoros), bugs, and lice. In the case of S. duttoni the spirochætes apparently break up in the body of the tick into very minute forms, and then pass into the egg, and so to the next generation of nymphs (see p. 236).

For the spirochætes of syphilis and yaws the name Treponema was proposed by Schaudinn;*^[4] the parasites are termed respectively T. pallidum and T. pertenue.

2. The Chlamydozoa

A name proposed by v. Prowazek to include a number of doubtful organisms, as the name implies. These problematic bodies are so minute that they are said to pass through a bacterial filter. They are parasites of epiblastic cells, but according to some authorities they merely represent a nuclear degeneration.

It is enough to mention that such diseases as vaccinia, variola, trachoma, epithelioma contagiosa of birds, verruga Peruana, measles, and foot-and-mouth disease of animals, have been ascribed to the agency of these Chlamydozoa.

1. * The terms "intermediary" and "definitive," as describing the vertebrate and invertebrate host, have often been used in a loose sense, thus leading to much confusion.
2. It has been suggested that the terms "flagellar" and "aflagellar" be used to designate the extremities of the body, instead of the terms "anterior" and "posterior," which are here employed strictly with reference to the mode of progression.
3. T. rhodesiense greatly resembles T. gambiense, but is more pleomorphic and more virulent to the lower animals.
4. * Schaudinn believed the spirochætes to be related to the trypanosomes. This conclusion was reached, it is believed, as the result of faulty observations on the blood-parasites of the little owl, which have already been referred to.