Chapter VI: Appendix: On Trematoda and Nematoda 753 (3)

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[10] (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.

[10] “Amœbic Dysentery,” _Johns Hopkins Hosp. Repts._, ii, pp. 395–548, 7 plates.

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[11] and then by
Casagrandi and Barbagallo,[12] 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.

[11] “On _Amœba blattae_,” _Proc. Acad. Nat. Sci._, Philadelphia (1879), xxxi, p. 204.

[12] “_Entamœba hominis_ s. _Amœba coli_ (Lösch).” _Annali d’Igiene speriment._ (1897), vii, p. 103. See also further remarks on p. 34.

*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. 1), 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. 2) 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. 4, 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)[13], 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.

[13] _Archiv f. Protistenkunde_, xxiv, p. 182.

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. 40).

According to Chatton and Lalung-Bonnaire[14] (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.

[14] _Bull. Soc. Path. Exotique_, v, p. 135.

*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. 6), 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, 8_a_) must be considered (see p. 38).

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.[15] 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[16]
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.

[15] Lung abscesses generally arise by the bursting of a liver abscess through the diaphragm into the right lower lobe of the lung, sometimes also through conveyance of amœbæ by means of the blood-stream (Banting).

[16] These findings were confirmed by Schaudinn by means of investigations on cats and men. _Cf._ also Alfred Gross, Marchoux, P. G. Woolley, W. E. Musgrave, H. F. Harris and others.

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[17] 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. 9, _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[18] 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. 41). 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.

[17] _Arb. a. d. kaiserl. Gesundheitsamte_, xix, pp, 547–576.

[18] “Life cycle of _Amœba coli_ in Human Body,” _American Medicine_, 1904, vii, p. 299; viii, p. 185.

_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. 5). 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,[19] lay stress on the internal structure of the nucleus (fig. 8, _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.

[19] _Arch. f. Protistenkunde_ (1911), xxiv, p. 163.

The diameter of the trophozoites or vegetative forms (fig. 8, _a_) is variously given as from 20 µ to 40 µ. Multiplication proceeds by binary fission and also by schizogony into four merozoites.[20]

[20] _See_ Darling, 1913, _Arch. Intern. Med._, vol. ii, pl. i, fig. 3.

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)[21] 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.

[21] _Bull. Soc. Med. et Chirurg. Indo-Chine_, iv, p. 474.

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_.[22]

[22] _Centralbl. f. Bakter._, Orig., lii, p. 335.

Walker,[23] Darling,[24] Wenyon[25] 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.

[23] _Philip. Journ. Sc._ (1911), B, vi, p. 259.

[24] _Annals Trop. Med. and Parasitol._ (1913), vii, p. 321.

[25] _Brit. Med. Journ._, Nov. 15, 1913, p. 1287, and _Journ. Lond. School Trop. Med._, ii, p. 27.

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[26] 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.

[26] Noc, F. (1909), _Ann. Inst. Pasteur_, xxiii, p. 177.

Certain other Entamœbæ[27] 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.

[27] See Fantham, H. B. (1911), _Annals Trop. Med. and Parasitol._, v, p. 111.

_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)[28] 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_).

[28] _Centralbl. f. Bakter._, Orig., lii, p. 335.

_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)[29] 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.

[29] _Journ. of Med. Research_, xxix, p. 43.

*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[30] (Feb. 1915) and Smith, Middleton and Barrett (1914) state that _E. buccalis_ is the cause of pyorrhœa alveolaris.

[30] _Journ. Amer. Med. Assoc._, lxiv, p. 553.

_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[31] (1911) considers that this parasite is probably identical with _Entamœba histolytica_.

[31] “The Parasitic Amœbæ of Man,” Lippincott, Philadelphia.

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.[32]

[32] See Craig (1913), _Amer. Journ. Trop. Dis. and Prevent. Med._, i, p. 351.

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[33] 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[34] (1910, 1914) described an entamœba, _E. mortinatalium_, from the lungs and other organs of infants in America.

[33] _Arch. de Parasitologie_, i, p. 275.

[34] _Amer. Journ. Trop. Dis. and Prevent. Med._, ii, p. 256.

*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.)