Class I. Sarcodina, Bütschli, 1882. Order. Amœbina, Ehrenberg. A. Human Intestinal Amœbæ

The first record of the occurrence of amœba-like organisms in the human intestine, that is, in intestinal evacuations, was that of Lambl (1859); nevertheless, the case was not quite conclusive, as the occurrence of testaceous amœbæ of fresh water (Arcella, Difflugia) was also reported. In 1870 Lewis found amœbæ associated with disorders of the large intestine in patients in Calcutta. A year later Cunningham reported from the same locality that he had observed on eighteen occasions, in one hundred examinations of dejecta from cholera patients, colourless bodies with amœboid movements, which became encysted and multiplied by fission. The daughter forms were said to be capable of dividing again, but they might also remain in contact. Contractile vacuoles were not noticed. The same bodies were observed also in simple diarrhœa (twenty-eight cases out of one hundred.)

The case reported by Lösch in 1875 attracted more attention. It was that of a peasant, aged 24, who came from the province of Archangel. He was admitted into Eichwald’s clinic at Petrograd with symptoms of dysentery. In the discharges containing blood and pus, Lösch found amœbæ in large numbers. When at rest these amœbæ measured from 20 µ to 35 µ; in a state of movement their length might extend up to 60 µ (fig. 1). The pseudopodia appeared only singly, and, since they were hyaline (ectoplasmic), were thus distinguished from the markedly granular endoplasm that enclosed a spherical nucleus of from 5 µ to 7 µ in diameter. One or more non-contractile vacuoles were present. Quinine enemata had the effect of making the amœbæ disappear from the fæces and thus causing the diarrhœa to abate. Four months after admission the patient died from the results of intercurrent pneumonia. At the autopsy ulceration of the large intestine was found, especially in the lower parts. Lösch connected the amœbæ with the ulcerations by experiments made on four dogs by injecting them with recently passed stools (per os et anum). Eight days after the last injection numerous amœbæ were found in the fæces of one of these dogs; eighteen days after the injection the animal was killed. The mucosa of the rectum was inflamed, covered with blood-stained mucus and ulcerated in three places. Numbers of amœbæ were found both in the pus of the ulcers and in the mucus. The three other dogs remained healthy. From these observations Lösch concluded that the species of amœba described by him as Amœba coli could not be regarded as the primary cause of the disease, but that it was certainly capable of increasing a lesion of the large intestine already present, or at least of preventing its healing.

B. Grassi (1879) found in the stools of healthy as well as in those of diarrhœic patients from various localities in Northern Italy, amœbæ similar to those discovered by Lösch. As this was of frequent occurrence, the pathogenicity could not be definitely established. Normand, formerly naval surgeon at Hong-Kong, observed numerous amœbæ in the dejecta of two patients suffering from colitis.

Many further investigations, which cannot be quoted in detail, showed not only that intestinal amœbæ were widely distributed in man, but indicated with greater certainty their rôle as agents of dysentery. The Commission sent out by the German Government in the year 1883 to investigate cholera in India and Egypt—whose members discovered the cholera bacillus—also collected information with regard to dysentery. In five cases of dysentery examined post mortem at Alexandria, with the exception of one case in which ulceration of the colon had already cicatrized or was approaching cicatrization, R. Koch found amœbæ as well as bacteria in sections from the base of the ulcers, although such had previously escaped notice in examination of the dejecta. Encouraged by these results, Kartulis (1885), who had discovered amœba-like bodies in the stools of patients suffering from intestinal complaints at Alexandria, continued his investigations. The results, obtained from more than 500 cases, gave rise to the theory that typical dysentery was caused by amœbæ as were also the liver-abscesses that often accompany it. Kartulis supported his theory not only by the regular occurrence of amœbæ in the stools of dysenteric patients and their absence in other diseases, and by the occurrence of the parasites in ulcers of the large intestine and in the pus from liver-abscesses, but also by experiments which he performed on cats. These were infected by injection per anum of stool material rich in amœbæ from subjects of dysentery. The infection took place also when amœba-containing, but bacteria-free, pus from liver-abscesses was used. It has been objected that the infection of man with Amœba coli, as the dysenteric amœbæ were then generally designated, does not take place per anum but per os. This difficulty, however, diminished in proportion as the encysted states of amœbæ (fig. 2), long known in the case of other Protozoa, became understood. The infection of man (Calandruccio, 1890) and of cats (Quincke and Roos) succeeded solely when material containing such stages was used. Amœbæ introduced into the intestine multiply there by fission (Harris, 1894). However, this theory, to which various other authors gave support on the grounds of their own observations, encountered opposition. Thus it was established that amœbæ were not found in patients in every place where dysentery was endemic, or else they were much rarer than was expected. Further, amœbæ were present in the most varied kinds of intestinal diseases, both of infective and non-infective characters. Also they were present in quite healthy persons.

Moreover, for various reasons, infection experiments on animals failed to supply proof, and finally a bacterium was discovered (Shiga, 1898) to be the excitant of one form of dysentery. Agglutination attested the specific part played by this organism, as it was produced by the blood serum of a person suffering from or recovered from dysentery, but not by the serum of one who was uninfected. Bacillary dysentery consequently was a distinct entity. The final step to be taken was to decide whether there was a specific amœbic enteritis (amœbic dysentery or amœbiasis, according to Musgrave).

This question should decidedly be regarded from the positive point of view. It is intimately connected with another, namely, whether there are not several species of intestinal amœbæ. The possibility of this had already been recognized. In addition to the Amœba coli Lösch, R. Blanchard distinguished yet another, Amœba intestinalis, and designated thereby the large amœbæ described in the first communication made by Kartulis; later on he stated the distinction between the species. Councilman and Lafleur (1891) considered the amœba of dysentery to be Amœba coli Lösch and so re-named the species Amœba dysenteriæ. Kruse and Pasquale (1893) employed the same nomenclature, but retained the old name Amœba coli Lösch for the non-infectious species. Quincke and Roos (1893) set forth three species: a smaller species (25 µ) finely granular, pathogenic for men and cats (Amœba coli Lösch); a larger species (40 µ) coarsely granular, pathogenic for men but not for cats (A. coli mitis); and a similar species non-pathogenic either for man or cat (A. intestini vulgaris). Celli and Fiocca (1894–6) went still further, they distinguished:

(1) Amœba lobosa variety guttula (= A. guttula Duj), variety oblonga (= A. oblonga Schm.) and variety coli (= A. coli Lösch).

(2) Amœba spinosa n. sp. occurring in the vagina as well as in the intestine of human patients suffering from diarrhœa and dysentery.

(3) Amœba diaphana n. sp. found in the human intestine in cases of dysentery.

(4) Amœba vermicularis Weisse, present in the vagina and in dysentery; and

(5) Amœba reticularis n. sp. in dysentery.

Shiga distinguished two species; a larger pathogenic species with a somewhat active movement, and a small harmless species with a somewhat sluggish movement. Bowman mentions two varieties, Strong and Musgrave (1900) two species—the pathogenic Amœba dysenteriæ and the non-pathogenic Amœba coli; Jäger (1902) and Jürgens (1902) mention at least two species. In the following year (1903) a work by Schaudinn was published which marked a real advance. This, in conjunction with the establishing of a special genus (Endamœba or Entamœba) for human intestinal amœbæ first by Leidy and then by Casagrandi and Barbagallo, for the time cleared up the confused nomenclature, the old name Amœba coli being retained for the harmless intestinal amœbæ of man, whereas the pathogenic species was designated Entamœba histolytica. The history of more recent work is incorporated in the accounts of the entamœbæ given below.

Entamœba coli, Lösch, 1875, emend. Schaudinn, 1903.

Syn.: Amœba coli, Lösch, 1875. Entamœba hominis, Casagr. et Barbag. 1897.

The amœboid trophozoite, according to Lösch, measures 26 µ to 30 µ and upwards; according to Grassi 8 µ to 22 µ; according to Schuberg 12 µ to 26 µ. A separation of the body substance into ectoplasm and endoplasm is only perceived during movement. The pseudopodia, which are generally only protruded singly, are broad and rounded at the end (lobopodia) and are hyaline, while the remainder of the body is granular. The ectoplasm is less refractile than the rest of the cytoplasm; it also stains less intensely (fig. ), and is best seen on protrusion of a pseudopodium. Red blood corpuscles are rarely, if ever, found ingested in the cytoplasm.

The nucleus is vesicular, and is spherical when inactive, measuring 5 µ to 7 µ, with a thick nuclear membrane. In the centre of the nucleus is a chromatinic body or karyosome or sometimes several small nuclear bodies formed of plastin and chromatin; the remaining chromatin is arranged on the achromatic network in the form of fine granules, especially thickly deposited on the nuclear membrane.

Entamœba coli lives as a commensal in the upper portion of the large intestine, where the fæces still possess a pulpy consistency. With their concentration and change in reaction lower in the bowel, the parasites either die or else if they are at a suitable stage of development form resistant cysts. These cysts (fig. ) can be found in great abundance in normal fæces, as Grassi first observed. Slight laxantia or intestinal diseases of any kind producing increased peristalsis, however, show amœbæ even in the unencysted condition, provided that the person harbours intestinal amœbæ generally. The intensity of infection varies according to the locality; thus Schaudinn found that 50 per cent. of the persons examined were infected with harmless amœbæ in East Prussia, 20 per cent. in Berlin and about 66 per cent. on the Austrian littoral.

The life-history (fig. 3) of the parasite exhibits two phases: (a) asexual multiplication in the intestine, either by binary fission or by schizogony with formation of eight merozoites, and (b) sporogony leading to the production of eight-nucleate cysts. Infection results from ingestion of cysts. Only cysts with eight nuclei are infective. The diameter of such cysts is about 15 µ to 20 µ.

There are varying accounts of the details of the life-cycle of Entamœba coli in its different stages. Thus, regarding schizogony or multiple fission it was formerly stated that the nucleus of the parent amœba divided into eight portions, which after dissolution of the nuclear membrane, passed outwards into the cytoplasm, which segregated around each. Eight merozoites were thus produced. More recently the process of schizogony has been considered to consist in the repeated division of the nucleus into two, four, and finally eight nuclei (fig. 3, A-D), and the formation of eight merozoites or amœbulæ.

The process of encystment is initiated by the extrusion of all liquid and foreign bodies from the protoplasm, which assumes a spherical form (fig. , A). The rounded uninucleate amœba then secretes a soft gelatinous coat, which finally differentiates into a double contoured cyst wall in older cysts. According to Casagrandi and Barbagallo, the size of the cyst varies from 8 µ to 30 µ, and averages about 15 µ. According to Schaudinn (1903) the cytological changes during cyst formation are as follows. The nucleus of a rounded uninucleate form divides into two (fig. 4, B). Each of these nuclei fragments into chromidia (fig. 4, C), some of which are absorbed, while others reunite so that the cell becomes binucleate again. Each of these nuclei, by a twice repeated division, produces three nuclei (fig. 4, D), the smaller two of which degenerate and were regarded as reduction nuclei. There is a clear zone or vacuole in the middle of the cyst during these maturation processes, dividing the cyst into two halves. After the nuclear reduction the clear space disappears, and each nucleus (termed by some a gamete nucleus) divides into two pronuclei (fig. 4, E). The pronuclei of the pairs were said by Schaudinn to differ slightly. Copulation occurs between pairs of unlike pronuclei, and is an example of autogamy (fig. 4, F). When complete, each of the fusion nuclei (synkarya) divides twice, giving rise first to four and finally to eight nuclei. Eight amœbulæ are thus formed within the cyst.

According to Hartmann and Whitmore (1911), however, autogamy does not occur within the cysts of E. coli. They consider that eight small amœbulæ are formed (fig. 3, 2-10) which escape from the cyst and then conjugate in pairs (fig. 3, 10-12), afterwards growing into a new generation of trophozoites.

Only some 10 to 20 per cent. of the cysts evacuated with the fæces undergo the full course of development, the majority perish previously. In old dry fæces, only cysts with eight nuclei are found, and it is these alone that cause the infection.

Entamœba williamsi, E. bütschlii, E. hartmanni and E. poleki (Prowazek) are probably only varieties of E. coli.

The principal feature distinguishing Entamœba coli from E. histolytica is the formation of eight-nucleate cysts by the former as contrasted with the tetra-nucleate cysts of the latter. The cyst-wall of E. coli is thicker than that of E. histolytica (tetragena). Further, E. coli does not usually ingest red blood corpuscles, nor are “chromidial blocks” present inside its cyst (see p. ).

According to Chatton and Lalung-Bonnaire (1912) the entamœbæ of vertebrates should be placed in a separate genus Löschia, as they differ in their life-history from E. blattæ, the type species of Entamœba. Leidy (1879), however, named the genus Endamœba, but further researches are necessary on biological variation among these organisms.

Entamœba histolytica, Schaudinn, 1903.

Syn.: Amœba coli, autt. p. p. Amœba dysenteriæ, autt. p. p.

The average size of the amœboid trophozoite is 25 µ to 30 µ. In fæces diluted with salt solution the amœbæ swell to 40 µ and more. There is sometimes separation of the body substance into a strongly refractile vitreous ectoplasm and a corneous endoplasm, pronounced even in repose, although the former is not equally thick at all parts of the periphery. In the endoplasm generally there are numerous foreign bodies (bacteria, epithelial cells, colourless and red blood corpuscles (fig. ), and occasionally living flagellates of the intestine). The nucleus is 4 µ to 6 µ in diameter, and may be difficult to recognize because it is sometimes weakly refractile and poor in chromatin. Its shape is slightly variable; it is usually excentric, sometimes wholly peripheral at the limit of the two parts of the body. Vacuoles are not present in quite fresh specimens, but appear later. In the study of E. histolytica, the morphological characters of the trophozoite or vegetative stage of the organism formerly separated as E. tetragena (figs. 5, 6, a) must be considered (see p. ).

The history of the development of these species, which give rise to amœbic enteritis as distinguished from bacillary dysentery, was formerly not so well known as that of E. coli. Upon being introduced into cats (per anum) dysenteric amœbæ provoke symptoms similar to those in man. In the latter, besides metastatic liver abscesses, abscesses of the lungs, and, according to Kartulis, cerebral abscesses are occasionally produced. Marchoux (1899) states that when the disease has lasted for some time liver abscesses are produced in cats also.

In the large intestine of infected cats the amœbæ creep over the epithelium, and here and there they force the epithelial cells apart, as well as removing them or pushing them in front of them; the amœbæ thus insert themselves into the narrowest fissures. They penetrate also into the glands through the epithelium, and thence into the connective tissue of the mucosa. Intestinal and glandular epithelia perish under the influence of these parasites: the cells are pushed aside, fall to pieces or are absorbed by the amœbæ. In the connective tissue of the mucosa the amœbæ migrate further, and often accumulate above the muscles. Finally they rupture this and force their way into the submucosa. In cats, apparently, the penetration is not so great as in men, according to Kruse and Pasquale. During their migration the parasites also gain access to the lymph-follicles of the wall of the intestine, which become swollen and commence to suppurate; follicular abscesses arise and after their rupture follicular ulcers. The diseased patches in the mucosa are markedly hyperæmic and numerous hæmorrhages are set up. Roos and Harris state that the amœbæ also penetrate into the blood-vessels (fig. 7) and this explains the occurrence of metastatic abscesses. The whole submucosa is severely swollen at the diseased spot and undergoes small-celled infiltration in the neighbourhood of the colonies of amœbæ. From these findings Jürgens (1902) draws the conclusion which is followed here, that the amœbæ are causative agents of the enteritis of cats, which disease is well defined, both pathologically and anatomically. Subsequent researches confirm the experience of earlier authors; great precautions were taken to exclude errors, hence, as with Gross and Harris, no exception can be taken to their results. The inoculation material was derived from soldiers who suffered from amœbic enteritis in China and who were admitted into the garrison hospital at Berlin. In order to be independent of the patients themselves, transmission experiments from cat to cat were performed, after the first experiments on cats yielded positive results. This was also effected by rectal feeding as employed by earlier workers. Such appeared necessary in order to prevent the evacuation of the inoculation material per anum, as well as to avoid the employment of morphia and ether narcosis. Forty-six cats were used for the experiments. Ten cats received tested stools containing motile amœbæ from soldiers suffering from amœbic enteritis contracted in China. Sixteen other cats received stools from cats infected by inoculation. All the animals sickened and suffered from the disease. Five cats received dejecta from human amœbic enteritis in which, however, no motile amœbæ were present. Thirteen cats received stools from soldiers who suffered from bacillary dysentery. None of the latter cats took the complaint and none showed changes in the large intestine upon sectioning. The injection of various bacteria, obtained from a stool of amœbic enteritis pathogenic to cats, remained without result in both the cats employed for this experiment. Lastly, two cats, which had been kept with those artificially infected, were taken ill spontaneously and suffered from the disease. In the opinion of Harris, who ascertained the harmless nature of bacteria derived from the intestinal flora containing dysenteric amœbæ, young dogs are capable of being infected.

Within the large intestine an active increase of Entamœba histolytica must occur. Nevertheless, Jürgens did not definitely find changes that might be interpreted in this sense. Schaudinn (1903) observed division and gemmation in vivo. Both processes, in which the nucleus divides by amitosis, can only be distinguished by the fact that the daughter individuals are similar in binary fission but dissimilar in gemmation, whether they make their appearance singly or in greater numbers. Schizogony, resulting in the formation of eight individuals, which is so characteristic for Entamœba coli, was not observed. (But schizogony, into four merozoites, is now known to occur. Gemmation processes are apparently degenerative.)

Resistant stages, which serve for transmission to other hosts, are according to Schaudinn first formed when the diseased portions commence to heal, or more accurately, the recovery commences when the vegetative increase of the amœbæ in the intestine discontinues. The so-called spores of E. histolytica were distinguished very definitely from those of E. coli; they were said to consist of spheres of only 3 to 7 µ in diameter, which were surrounded by a double membrane, at first colourless, but becoming a light brownish yellow colour after a few hours, and possessing a protoplasmic content containing chromidia. They were said to arise by fragments of chromatin passing outwards from the nucleus of the amœba into the surrounding cytoplasm (fig. , a) and undergoing so marked an increase that finally the whole cytoplasm became filled with chromidia. The remainder of the nucleus underwent degeneration and became extruded. On the surface of the cytoplasm there then arose small protuberances containing chromidia. These processes had been observed in the living organisms. They gradually divided and separated from membranes which later became yellow. The remainder of the amœba perished. Craig had also seen phases of this process of development. It must be remarked that, according to recent researches, these processes of exogenous sporulation are degenerative in character (see p. ). The small spores may be fungi. The “sporulation” processes are only mentioned here as a warning. They are now only of historic interest. By means of an experiment made on a cat, Schaudinn ascertained that ingestion of permanent cysts, which resist desiccation, is the cause of the infection. The animal took food containing dry fæces with amœba cysts; these fæces came from a patient suffering from amœbic enteritis in China. On the evening of the third day the cat evacuated blood-stained mucous fæces which contained large numbers of typical Entamœba histolytica. On the fourth day after the infection the animal experimented upon died, and the large intestine showed the changes previously stated.

E. histolytica also is found in the large intestine. This was originally shown to be the case by Kartulis, and the fact has recently been confirmed from many quarters. It is also present in the metastatic abscesses of which it is the cause (cf. among other authors, Rogers, Brit. Med. Journ., 1902, ii, No. 2,177, p. 844; and 1903, i, No. 2,214, p. 1315).

It should lastly be pointed out in this connection that mixed infections also take place. For instance, in addition to E. histolytica, E. coli, and, under certain circumstances, flagellates may be found together. In the same way E. coli may come under observation even in bacillary dysentery. On the other hand, Schaudinn stated that in cases of dysentery endemic in Istria, Entamœba coli, if it had hitherto been present, disappeared, to return again after recovery from the illness.

(Entamœba tetragena, Viereck, 1907.)

This amœba must now be considered to be a part of the lifecycle of Entamœba histolytica, in fact a very important part of that cycle, especially in its tetranucleate cystic stages.

This organism, the so-called Entamœba tetragena, may occur in the human intestine in cases of amœbic dysentery, especially in mild or chronic cases. It was discovered by Viereck in 1907 in patients suffering from dysentery contracted in Africa. Soon afterwards an independent description was published by Hartmann, who called the amœba E. africana. It was also studied by Bensen and Werner. Recently (1912–13) much work has been published on this amœba by Darling and others; in this way its relationship to Schaudinn’s E. histolytica has been made known.

In general morphology it somewhat resembles Entamœba coli, and its discoverer at first mistook it for a variety of that species. According to Hartmann, a distinct ectoplasm is only clearly visible when a pseudopodium is protruded (fig. ). The granular endoplasm may contain ingested red blood corpuscles (fig. 6). The large, round nucleus is visible in the fresh state (fig. 8, a). So-called chromidial masses (? crystalloidal substances) may occur in the cytoplasm.

Some investigators, as Hartmann, lay stress on the internal structure of the nucleus (fig. , b, c), best seen in preparations fixed wet and stained with iron-hæmatoxylin. The nucleus is limited by a well-marked nuclear membrane, on the inside of which granules or nodules of chromatin may occur. There is a karyosome, which, in successfully stained specimens, shows, at times, a central dot called a centriole. (The nucleus of Entamœba coli does not contain such a centriole.) However, the structure of the nucleus varies at different periods during the life-cycle.

The diameter of the trophozoites or vegetative forms (fig. , a) is variously given as from 20 µ to 40 µ. Multiplication proceeds by binary fission and also by schizogony into four merozoites.

Reproduction takes place by endogenous encystment (fig. 9, d-f), which is preceded by nuclear division into two, reduction and then autogamy. The interpretation of the latter phenomenon as autogamy is disputed by some authors. The round cysts, which may measure 12 µ to 15 µ in diameter, contain four nuclei, together with darkly staining masses of various shapes, the so-called “chromidial blocks” (fig. 9, f). The cyst-wall of E. histolytica (tetragena) is thinner than that of E. coli, and the diameter of the cyst is rather less. E. histolytica has not yet been cultivated.

Infection in man occurs by way of the mouth by the ingestion of cysts. A patient showing acute symptoms of dysentery is not usually infective, for he is merely harbouring the large trophozoites, which, by experiment, have been shown not to be infective to animals (kittens) when administered by the mouth. The stools of recovered patients may still contain cysts, and they may thus act as cyst-carriers or reservoirs of disease by infecting water and soil. The stools of such cyst-carriers are often solid, and so cysts of E. histolytica (tetragena) are easily overlooked. Mathis (1913) points out that healthy carriers of E. histolytica may be found; 8 per cent. of the natives of Tonkin examined by him were healthy carriers of cysts.

In return cases, or prolonged untreated cases of entamœbic dysentery, a generation of smaller trophozoites is associated with, or replaces the larger ones. In stools they are frequently refractile and consequently stain slowly intra vitam. These trophozoites are the “smaller, senile, or pre-cyst generation” of Darling. This pre-cyst generation is characterized by the presence of blocks of crystalloidal substance in the cytoplasm, and by the possession of a prominent, densely stainable karyosome. Darling believes this generation to be the same as that described by Elmassian as Entamœba minuta.

Walker, Darling, Wenyon and others believe that Entamœba histolytica, which was only seen by Schaudinn in a single case, that of a Chinaman, is really E. tetragena. Darling states that if the published illustrations of E. histolytica and of E. tetragena are collected from the literature and compared, it will be seen that the writers have been calling E. histolytica the large trophozoites seen in dysenteric stools. These large trophozoites frequently display no karyosome, but they can be demonstrated as E. tetragena by animal inoculation, or by the history of the case. On the other hand, the illustrations of E. tetragena show that the authors have been dealing with the small generation or reduced forms (“E. minuta”), which are the direct descendants of the large trophozoites. If kittens are inoculated rectally with dysenteric material containing large trophozoites, the strain may be carried in successive kittens for four to six transfers. If, on the other hand, kittens are inoculated rectally with small trophozoites of the pre-cyst generation, the transmission cannot be carried through more than one or two kittens. Wenyon has succeeded in maintaining E. tetragena in kittens for several generations.

In some of the preparations from the last remove, pathological forms of the trophozoites may be seen. These show abnormal forms of budding, especially peripherally, such as have been described by Schaudinn and by Craig as characteristic of E. histolytica. Schaudinn’s small peripheral, exogenous buds and cysts are thus explained. Craig has latterly changed his views.

Further, Darling states that tetragena cysts fed by the mouth to kittens produce bowel lesions in which trophozoites having the characters of E. tetragena, E. histolytica and E. nipponica (Koidzumi) occur.

In view of the work of recent observers, the peculiar exogenous encystment which Schaudinn made characteristic of Entamœba histolytica has been shown to be due to degenerative changes in senile races of the amœba. E. histolytica and E. tetragena are one and the same species, and its trophozoite is subject to variation. According to some observers the histolytica type of nucleus—described by Schaudinn as being poor in chromatin and not easily seen in the fresh state—occurs frequently in patients with severe symptoms of dysentery; on the other hand, the tetragena type of nucleus—round and easily seen in the fresh state—may occur in cases presenting slight dysenteric symptoms. Intermediate types of nuclei are seen. The name of this species, the principal pathogenic amœba of man, must then be E. histolytica by priority. The cystic stages of E. histolytica are those first recorded by Viereck and formerly described as E. tetragena. The geographical distribution of E. histolytica is wide.

Noc’s Entamœba (1909).

A species of Entamœba was cultivated by Noc in 1909 from cysts derived from liver abscesses, from dysenteric stools and from the water supply of Saigon, Cochin China. He cultivated it in association with bacteria. It is pathogenic. It has been considered allied to E. histolytica, and shows internal segmentation or schizogony. It exhibits polymorphism. This amœba has been found by Greig and Wells (1911) in cases of dysentery in India. It is an important organism and requires further investigation.

Certain other Entamœbæ have been described at various times from the intestinal tract of man. Probably most, if not all, of these are not good species and in some cases much more information is needed.

Entamœba tropicalis (Lesage, 1908). This parasite is said to be non-pathogenic, and to occur in the intestine of man in the tropics. It has a general resemblance to E. coli, but forms small cysts (6 µ to 10 µ in diameter). The nucleus of the cyst is said to break up into a variable number of daughter nuclei, from three to thirteen having been noted. Lesage states that it is culturable in symbiosis with bacteria. It is probably a variety of E. coli, if not a cultural amœba.

Entamœba hominis (Walker, 1908) has a diameter of 6 µ to 15 µ. A contractile vacuole is present. Encystment is total, and small cysts are formed. It is culturable. The original strain, now lost, was obtained from an autopsy in Boston Hospital. This organism is probably a cultural amœba.

Entamœba phagocytoides (Gauducheau, 1908). This parasite was discovered in a case of dysentery at Hanoi, Indo-China. The amœba is small, 2 µ to 15 µ in diameter. It is active. It ingests bacteria and red blood corpuscles, while peculiar spirilla-like bodies are found in its cytoplasm. It multiplies by binary and multiple fission. It can be cultivated. More recently (1912) the author appears to consider the amœba to be a stage of a Trichomonas, but abandons the view later (1914). Further researches on this organism are needed.

Entamœba minuta (Elmassian, 1909) was found, in association with E. coli, in a case of chronic dysentery in Paraguay. It resembles E. tetragena but is smaller, rarely exceeding 14 µ in diameter. Schizogony occurs, four merozoites being produced. The encystment is total and endogenous, giving rise to cysts containing four nuclei. This amœba is considered by Darling and others to be the pre-cyst trophozoite stage of E. histolytica (tetragena).

Entamœba nipponica (Koidzumi, 1909) was found in the motions of Japanese suffering from dysentery or from diarrhœa, in the former case in company with Entamœba histolytica. Its diameter is 15 µ to 30 µ. The endoplasm is phagocytic for red blood corpuscles. The nucleus is well defined, resembling that of E. coli and of E. tetragena. Multiplication occurs by binary fission and by schizogony. Encystment is total, but has not been completely followed. Darling and others consider that this is an abnormal form of E. histolytica, while Akashi (1913) doubts if it is an amœba at all, but rather is to be regarded as shed epithelial cells.

General Remark.—It is now considered by some workers that true Entamœbæ cannot be cultivated on artificial media. Quite recently Williams and Calkins (1913) have somewhat doubted this opinion, and state that certain cultural amœbæ, originally obtained from Musgrave in Manila, exhibit the various morphological variations associated with true entamœbæ of the human digestive tract.

Entamœba buccalis, Prowazek, 1904.

The size varies from 6 µ to 32 µ. Ectoplasm is always present; the endoplasm contains numerous food-vacuoles. The nucleus is vesicular, with a greenish tinted membrane which is poor in chromatin. The size of the nucleus is from 1·5 µ to 4·5 µ. A contractile vacuole is not visible. The pseudopodium is broad. It was discovered in the mouths of persons with dental caries at Rovigno and also at Trieste, being most easily found in dense masses of leucocytes, also among leptothrix and spirochæte clusters. It can be easily distinguished from leucocytes by more intense staining with neutral red. Multiplication proceeds by fission. Transmission may take place through the small spherical cysts. This species (fig. 10) has since been observed in Berlin, and is also occasionally found in carcinoma of various regions of the oral cavity. (Leyden and Löwenthal, 1905).

Entamœba buccalis, Prow., is said to be allied to a protozoön which A. Tietze has found either encysted or free in the lumen of the orifice of the parotid gland of an infant aged 4 months. The gland had undergone pathological change, and had therefore been extirpated. The organisms, which were roundish and three to four times the size of the normal epithelial cells of the gland, were without a membrane and possessed a nucleus in which the chromatic substance appeared to be contained in a karyosome. Bass and John’s (Feb. 1915) and Smith, Middleton and Barrett (1914) state that E. buccalis is the cause of pyorrhœa alveolaris.

Entamœba undulans, Aldo Castellani, 1905.

Under this name a protozoön is described which A. Castellani found in addition to Entamœba histolytica and Trichomonas intestinalis in the fæces of an European planter living in Ceylon, who had suffered from amœbic enteritis and liver abscess. The shape of the body was roundish or oval, 25 µ to 30 µ in the greatest diameter. It was without a flagellum, but with an undulating membrane, and capable of protruding a long pseudopodium from different parts of its body at short intervals. The nucleus could not always be recognized in life; it was, however, always demonstrable by staining. One or two contractile vacuoles were present. The protoplasm was finely granular, showing no differentiation into ecto- and endo-plasm. According to Braun, in spite of the author declaring himself expressly against the flagellate nature of the parasite, such a nature may be assumed to be tolerably certain in view of the description and illustration.

It is now considered that Entamœba undulans is a portion of a flagellate, namely, Trichomonas.

Entamœba kartulisi, Doflein, 1901.

Doflein gave this name to amœbæ, from 30 µ to 38 µ in diameter, which Kartulis (1893) found on examining the pus of an abscess in the right lower jaw of an Arab, aged 43, and in a portion of bone that had been extracted. The movements of the amœbæ (fig. 11) were more active than those of “dysenteric amœbæ.” Their coarsely granular cytoplasm contained blood and pus corpuscles, and a nucleus was generally only recognizable after staining. Vacuoles were not seen with certainty. Flexner reported upon a similar case, and Kartulis published five additional cases. As in these cases dental caries was present the infection is likely to have proceeded from the oral cavity as a result of the carious teeth. Craig (1911) considers that this parasite is probably identical with Entamœba histolytica.

In the literature the following species have been reported as occurring in the oral cavity of man:—

Amœba gingivalis, Gros, 1849. [? identical with Entamœba buccalis.]
Amœba buccalis, Sternberg, 1862.
Amœba dentalis, Grassi, 1879.

Far too little, however, is known concerning these to regard them as definite species, that is, independent organisms; Grassi thinks it even possible there may have been a confusion in their case with salivary corpuscles. If they really are amœbæ they are all of them probably identical with Entamœba buccalis.

Genus Paramœba, Schaudinn, 1896.

Schaudinn established the genus Paramœba for a marine rhizopod which multiplied by division, became encysted at the end of its vegetative life and then segmented into swarm bodies with two flagella. These multiplied by longitudinal fission, and finally passed into the condition of Amœbæ. Whether the human parasite described by C. F. Craig (1906) as

Paramœba hominis.

belonged to this genus was for a time uncertain. It is now placed in a new genus Craigia, Calkins, 1912, since it possesses only one flagellum.

In the amœbic stage it is 15 µ to 25 µ in diameter; ecto- and endo-plasm during rest are indistinguishable. The body substance is granular, with a spherical, sharply contoured nucleus and an accessory nuclear body. No vacuoles are present, but occasionally the endoplasm contains red blood corpuscles. The pseudopodia are hyaline, finger- or lobe-shaped, and are protruded either singly or in twos. Multiplication is by binary fission and by the formation of spherical cysts (15 µ to 20 µ in diameter) in which occurs successive division of the nuclei, ultimately forming ten to twelve roundish bodies each of which soon develops a flagellum. The flagellate stages have similarly a spherical shape and attain a diameter of 10 µ to 15 µ. They also occasionally contain red blood corpuscles and pass either directly or after longitudinal division into the amœboid phase.

Craig found these Amœbæ and the flagellate stage belonging to them in six patients in the military hospital at Manila (Philippine Islands), five of whom were suffering from simple diarrhœa whilst the sixth exhibited an amœbic enteritis and contained also Paramœba hominis, with Entamœba histolytica, Schaudinn. In one of the other cases, Trichomonas intestinalis was present.

B. Amœbæ from other Organs.

Entamœba pulmonalis, Artault, 1898.

Artault discovered a few amœboid forms with nucleus and vacuole in the contents of a lung cavity. In the fresh condition they were distinguishable from leucocytes by their remarkable capacity of light refraction. They were also much slower than the latter in staining with methylene blue or fuchsine. Their movements became more lively in a strong light. Water and other reagents killed them, and then, even when stained, they could not be distinguished from leucocytes. They have also been seen by Brumpt. R. Blanchard found amœbæ which may belong here in the lungs of sheep. A. pulmonalis is perhaps the same as Entamœba buccalis. Smith and Weidman (1910, 1914) described an entamœba, E. mortinatalium, from the lungs and other organs of infants in America.

Amœba urogenitalis, Baelz, 1883.

This species was found in masses in the sanguineous urine as well as in the vagina of a patient in Japan, aged 23. Shortly before the death of the patient, which was caused by pulmonary tuberculosis, hæmaturia with severe tenesmus of the bladder had set in. The amœba, which showed great motility, and had a diameter of about 50 µ when quiescent, exhibited a granular cytoplasm and a vesicular nucleus. Baelz is of opinion that these parasites were introduced into the vulva with the water used for washing the parts, and thence had penetrated into the bladder and vagina. Doflein places the organism in the genus Entamœba, and it is perhaps identical with E. histolytica.

Similar cases are also reported (1892–3) by other authors: Jürgens, Kartulis, Posner, and Wijnhoff. Jürgens found small mucous cysts, filled with amœboid bodies, in the bladder of an old woman suffering from chronic cystitis; they were also found in the vagina. The amœba observed by Kartulis in the sanguineous urine of a woman, aged 58, suffering from a tumour of the bladder, measured 12 µ to 20 µ, and exhibited slow movements by protruding short pseudopodia. The vacuoles and nucleus became visible only after staining with methylene blue.

Posner’s case related to a man, aged 37, who had hitherto been quite healthy and had never been out of Berlin. Suddenly, after a rigor, he passed urine tinged with blood. This contained, besides red and white blood corpuscles and hyaline and granular casts, large granular bodies (about 50 µ in length and 28 µ in breadth), which slowly altered their shape, and contained red blood corpuscles in addition to other foreign matter. These bodies exhibited one or several nuclei and some vacuoles. From the course of the disease, which extended over a year, and during which similar attacks recurred, Posner came to the conclusion that the amœbæ which had originally invaded the bladder had penetrated into the pelvis of the kidney, where they probably had settled in a cyst, and thence induced the repeated attacks.

Wijnhoff observed four cases of amœburia in Utrecht.

Amœba miurai, Ijima, 1898.

Under this term the author describes protoplasmic bodies which Miura, in Tokyo, found in the serous fluid of a woman, aged 26, who had died from pleuritis and peritonitis endotheliomatosa. Two days before death these same forms had also appeared in the hæmorrhagic fæces of the patient. The bodies were usually spherical or ellipsoidal, and at one pole carried a small protuberance (fig. 12) beset with filamentous short “pseudopodia” (really a pseudopodium covered with cilia). Their size varied between 15 µ and 38 µ. The cytoplasm was finely granular, and no difference was observable in the ecto- and endo-plasm, only the villous appendage was clearer. The cytoplasm contained vacuoles more or less numerous, none of which was contractile. After the addition of acetic acid one to three nuclei could be distinguished, 8 µ to 15 µ in size. Actual movements were not observed. Taking everything into consideration, the independent nature of these bodies is, to say the least, doubtful, although it cannot be denied that they possess a certain similarity to the marine Amœba fluida, Grüber or Greeff, and to a few other species. (It is likely that cells present in serous exudation were mistaken for amœbæ.)

Appendix.

“Rhizopods in Poliomyelitis acuta.”

In three cases of poliomyelitis acuta which were investigated by Ellermann, the spinal fluid obtained by puncture of the cord contained bodies, from 10 µ to 15 µ in size, which had amœboid movements and exhibited variously shaped pseudopodia in large numbers. After staining, a usually excentric nucleus, about 1·5 µ in size, was demonstrated in them.

Order. Foraminifera, d’Orbigny.

The order is divided by Max Schultze into Monothalamia and Polythalamia. Only a few of the former can be considered here.

Sub-Order. Monothalamia. (Testaceous Amœbæ).

These forms occur frequently in fresh water, rarely in sea water. They possess a shell which is either pseudo-chitinous in character, or consists of foreign particles, or in a few cases is composed of siliceous lamellæ. There is usually an orifice for the protrusion of pseudopodia. The only representative of the order of interest here is:—

Genus. Chlamydophrys, Cienkowski, 1876.

The genus is based on a form which A. Schneider carefully investigated and considered to be the Difflugia enchelys of Ehrenberg. L. Cienkowski rediscovered this same form and created for it the genus Chlamydophrys. We agree with this view, but not with the renaming of the organism (so common at the time). If the parasite in dung, Chlamydophrys stercorea Cienk. is identical with Difflugia enchelys of Ehrenberg, the old specific name should be retained.

The genus is characterized by the possession of a hyaline, structureless, slightly flexible shell which is ovoid or reniform. At the more pointed pole there is an orifice situated terminally or somewhat laterally, serving for the emergence of the filiform pseudopodia (fig. , a). The protoplasm does not entirely fill the interior of the shell. An equatorial zone bearing excretory granules divides the shell internally into two almost equal portions. The anterior portion is rich in vacuoles and serves for the reception of nutriment and for digestion. The posterior part is vitreous, and contains the nucleus. One to three contractile vacuoles are situated in the equatorial zone.

Chlamydophrys enchelys, Ehrbg.

Syn.: Chlamydophrys stercorea, L. Cienkowski.

This species (fig. 13) is found in the fæces of various animals (cattle, rabbits, mice, and lizards), and also in quite fresh human fæces. According to Schaudinn, the parasite occurs so frequently in the human fæces that it must be considered of wide distribution. The species must traverse the intestine of man and animals during one stage of its life cycle, as Schaudinn showed by experiments on himself and on mice. He infected himself with cysts (fig. ) by swallowing them, and evacuated the first Chlamydophrys as early as the following day. After the evacuation of numerous specimens on one of the following days the infection ceased.

The nucleus of a living specimen is surrounded by a hyaline, strongly refractile chromidial mass, arranged in the form of a ring. Chromatin stains colour it darkly.

Asexual multiplication (fig. 13, b), which takes place in fæces, follows a similar course to that of allied forms (e.g., Euglypha, Centropyxis). It commences by the cytoplasm issuing from the orifice of the shell and assuming the shape characteristic of the mother organism, but in a reverse position. The nucleus then divides by mitosis, when the daughter nuclei move apart from one another. The chromidial ring also divides into two portions by a process of dumb-bell like constriction. The one daughter nucleus remains in the mother organism, the other moves towards the daughter individual, which then separates from the parent.

In this species plasmogamic union of two or more individuals (up to twenty) is frequently observed. Such colonies may similarly divide, and in this way monstrosities frequently arise. When drying of the fæces, or deficiency of food occurs, encystment takes place apparently spontaneously. The whole body, as stated by Cienkowski, issues from the shell, assumes a spherical shape (probably with discharge of water) and becomes surrounded with a thick membrane (fig. 14). After the addition of water and the escape of the encysted Chlamydophrys, a new shell must be formed. Schaudinn, who has not given a more detailed description of the process of encystment in this species, but refers to Cienkowski and to similar observations made on Centropyxis, states of the latter that the encystment takes place within the shell.

The sexual multiplication is accompanied by shedding of all the foreign bodies and of the degenerating nucleus. The protoplasm, now contracting into a sphere, remains behind in the shell with the chromidial mass. From the latter several new nuclei arise (sexual nuclei) often eight in number. The cytoplasmic sphere then segregates into as many spherical portions as there are nuclei present. When they have assumed an oval form, two flagella develop at one pole and the flagellispores swarm out of the shell. The biflagellate swarm-spores, or gametes, copulate in pairs and apparently the individuals of the pairs of gametes arise from different mother organisms. The zygote secretes a thick covering which soon becomes brown and rough. These zygote cysts or resistant spores must now pass from the intestine of an animal in order to complete their development. The escape of the cyst contents does not always take place in the intestine; often it does not occur until after defæcation. These shell-less individuals (amœbulæ) soon become invested with a shell. But in the alkaline intestinal contents, shell formation may proceed even while the organism is in the intestine, and multiplication may take place.

Schaudinn’s further communication was of special interest; it was to the effect that Chlamydophrys was related to

Leydenia gemmipara, Schaudinn, 1896.

In the fluid removed by puncture from two patients suffering from ascites in the first medical clinic in Berlin, cellular bodies with spontaneous movement were found, which Leyden and Schaudinn regard as distinct organisms. They remained alive without the use of the warm stage for four or five hours, the external temperature being 24° to 25° C. In a quiescent condition they were of a spherical or irregular polygonal form. Their surface was rarely smooth, being beset with protuberances and excrescences (fig. ). The substance of the body was thickly permeated with light refractile granules with a yellowish shimmer. The hyaline ectoplasm was rarely seen distinctly. All sizes from 3 µ to 36 µ in diameter were observed. The movements were rather sluggish, the ectoplasm in the meantime appearing in the form of one or several lamellæ, in which also strings of the granular endoplasm occurred, and frequently protruded over the border of the hyaline pseudopodia. The tendency for the joining of several individuals by means of their pseudopodia was so marked that associations ensued similar to those known in free-living Rhizopoda.

The cytoplasm enclosed blood corpuscles as well as numerous vacuoles, one of which pulsated slowly about every quarter of an hour. A vesicular nucleus the diameter of which was about equal to one-fifth of the body was present.

Multiplication took place by means of division and budding (fig. 15, c), after previous direct division of the nucleus. The buds were supposed to divide repeatedly soon after their appearance, thus giving rise to minute forms of 3 µ.

There was a suspicion in both cases that the ascites was associated with malignant neoplasms in the abdomen, and autopsy confirmed this view in one case.

The parasite, which has seldom been observed, has been variously interpreted; for example, it has been regarded merely as altered tissue cells. It is now known, from Schaudinn’s researches, that Leydenia gemmipara is connected with abnormal conditions of Chlamydophrys, occasionally occurring as a commensal in the ascitic fluid. The form is produced when pathological conditions of the large intestine create an alkaline reaction of its whole contents. The formation of shells then often ceases, and these naked Chlamydophrys are enabled to multiply atypically by division and gemmation. Such stages, which are no longer capable of a normal development, are the Leydenia, as Schaudinn has demonstrated.