NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
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found in mammals and the appearance of the dog parasite is especially striking.
This is especially the case in specimens stained in a routine manner
with Romanowski's stain for, owing to the refusal of the parasite to stain it
is often seen while all else in the specimen is perhaps deeply coloured, only
as beautifully regular oval brilliantly transparent clear spaces in the leucocytes,
or as bodies faintly tinged with blue lying in this situation in their clear cyst-like
capsules.
The resistance of L. canis to staining is undoubtedly due to the capsule. The
capsule, according to James, is formed by a condensation of the protoplasm of
the leucocyte about the parasite. In my specimens this appearance of conden-
sation is very marked, the parasite being surrounded by a line of more deeply
staining protoplasm, which in some specimens appears to consist of closely
aggregated granules. This line on its outer edge merges imperceptibly into
the surrounding protoplasm of the leucocyte, but, on its inner edge, ceases so
abruptly against a clear space as to suggest that the parasite, in addition to a
layer of altered protoplasm, is surroun ded by a capsule proper to itself. Several
considerations support such a view. Not only is the edge of the oval space very
defined, but the contour is extremely regular, and the capsule, though described
as oval, is sufficiently flattened at the poles to give the impression of living form,
rather than merely of a space distended with fluid. It is significant, too, that the
contained parasite, however bulky, never quite touches the condensed proto-
plasm of the leucocyte, but leaves between itself and this a clear even line.
In stained films one not infrequently encounters forms quite free in the
plasma, and these possess the clear oval capsule, though they are unsupported
by any apparent cell substance.
The appearances in fresh preparations, especially when a trace of methyl
violet is present, are particularly suggestive of a true capsule, for the encapsuled
parasite appears as a solid and even rigid foreign body, often piercing the leuco-
cyte from side to side.
Perhaps the strongest reason of all for believing that the capsule of L. canis
is a true cyst, is that in the leucocyte parasite of the squirrel, though a
cytocyst is present, none of the above appearances are seen. There are
also forms of L. canis to be described later, which, though surrounded by
a layer of condensed protoplasm, stain readily.
The hæmogregarines of the red cells, H. gerbilli and H. balfouri, merely lie
in a space in the red cell and though in H. gerbilli the red cell may, in its altered
state, stain so that it resembles a thin enveloping membrane, there is no trace
of any proper capsule.
Within the capsule, in properly stained specimens, is seen the quadrangular,
bean-shaped, or vermicule-like parasite. The ordinary forms, so far as I have
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been able to ascertain, are without any trace of the 'tail' so characteristic of
H. gerbilli. The chromatin mass, which lies close to one end of the parasite,
stretches almost if not quite across the body of the parasite which is here at its
narrowest.
The chromatin mass is like that of H. gerbilli composed mainly of strands,
which pass transversely to the body of the parasite forming a rather open coarse
reticulum. The protoplasm is finely reticular and near the thicker extremity
of the parasite is often vacuolated. Owing to the position of the chromatin mass
at one end of the parasite the protoplasm is as a rule almost all collected in
front of this body. But there are forms in which the chromatin mass is more
centrally situated and the protoplasm more evenly divided.
The protoplasm may or may not exhibit granules. The number and
appearance of the granules varies with the stain employed and the depth of stain-
ing achieved. When weak Nocht's Romanow skis stain is used, the protoplasm
is blue and reticular, devoid in most cases of any granules. In specimens
coloured with Giemsa's stain fine reddish speckling is generally visible, and a few
small more distinct granules are often present. Novy's modification of Roma-
nowski's stain brings out numerous and quite large granules.
The invaded cells are said by James to be polymorphonuclear leucocytes,
and, after the examination of some of James' preparations which he was kind
enough to send me, I was myself convinced that it was this kind of cell that was
invaded. But further work on the subject and especially comparison of cells
containing L. canis with those invaded by L. funambuli has led me to modify my
opinion. It is some time since I saw these preparations of James', and I have
unfortunately not been able to re-study them, but considering the matter from
my new point of view, I have been struck by the fact that, though James depicts
in fig. 5 of his plate an invaded cell in which the nucleus differs from that of a
polymorphonuclear leucocyte only in being more opened out, all his other figures,
with one doubtful exception, represent cells the nuclei of which are not obviously
polymorphonuclear in nature.
As a matter of fact the great majority of cells in which parasites occur do not
conform exactly to any normal type of leucocyte. The nucleus stains more
diffusely and often more intensely, than the nuclei of other leucocytes. Most
commonly two masses of nuclear matter of which one is usually larger than the
other, are seen lying on each side of the parasite (fig. 2, a, d). Such a nucleus
is apt to give rise to different appearances which depend a good deal upon the
amount of flattening the cell has undergone in the making of the film. Very
often the nucleus is split up into a number of separate pieces, a condition which
seems to have resulted from the parasite pushing its way through and through this
structure, since every stage between slight indentation of the nucleus by the
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parasite and the condition where the nucleus is completely broken up, can be
made out. This fragmented condition of the nucleus is almost exactly reproduced
in mononuclear leucocytes of the squirrel which have been invaded by L. funam-
buli. The protoplasm of invaded cells, though it may contain a few fine granules
resembling those in the polymorphonuclear cells, is usually pale and devoid of
granules, and the outline of the cells is indistinct and often difficult to follow in
its entirety.
In fresh films invaded cells exhibit amœboid movement, but in most cases it
is quite clear that the cells are crippled by the presence of the parasite, for their
movements lack the vigour of those of healthy leucocytes, and in the time that a
normal cell will have traversed several fields of the microscope, they will scarcely
have moved from their original position.
As previously stated, these cells are unlike any normal type of leucocyte ;
even after much work I was at first unable, by direct observation, to be quite
sure what normal element the invaded cell did actually represent. This un-
certainty was increased by the fact that cells containing L. funambuli were
often identical in appearance with those invaded by L. canis. It was only as
a result of observations on the bone marrow that a satisfactory solution of this
question was found. As will be seen later, the cell in the peripheral blood
is a cell which invaded in its mononuclear stage in the bone marrow, has
developed towards a transitional stage, and possibly even may have reached a
polymorphonuclear stage, though modified by the destructive influence of the
parasite acting throughout its whole life period.
The existence of a motile vermicule stage in L. canis is suggested by the
single observation of James. This stage I have since been able to study both
in fresh and in stained films. In fresh preparations of the blood free
vermicule forms are seldom seen, L. canis in this respect offering a marked
contrast to H. gerbilli and to L. funambuli in both of which vermicules are
easily found in ordinary fresh blood preparations.
Though rare in vitro, the vermicules of L. canis occur in large numbers
and are readily studied in the gut of dog ticks ( R. Sanguineus) which have fed
on infected dogs. Seen in the gut of the tick, the vermicules exhibit active
movements. Very characteristic is a movement which consists in a gradual
flexion of the body, thus approximating the two extremities of the parasite
followed by a rather sudden movement of extension. The majority of vermi-
cules have one extremity much more pointed than the other, but stouter, more
sausage-shaped forms are also occasionally seen, exhibiting a characteristic
rotatory movement, which may be transmitted to masses of blood corpuscles
or other debris. In stained films the vermicules show a sharp and a blunt
extremity. The sharp end is transparent and free from granules ; the blunt
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end is more granular and stains more darkly. In the centre of the parasite is an
oval chromatin mass, which stains intensely. As a rule the chromatin mass is
more compact than it is in the encapsuled forms, but vermicules with a chro-
matin mass identical in appearance with that of the encapsuled parasite are
quite frequently seen. Even in stained films the tendency to lateral flexion is
shown by the curved position which the vermicules have adopted. (Fig, 3 a.)
Encapsuled forms are very rarely found in the tick, and the vermicules
are so abundant as to suggest that almost all the parasites here take the form
of active vermicules.
The function of the vermicules of the hæmogregarinidae is not yet altogether
clear. Hintze (8) has shown that in Lankesterella vermicules may perform sexual
functions. In H. clamatae (Stebbins) the parasite uses a free motile stage to
pass from cell to cell apparently for nutritive purposes only. The liberation of
vermicules on the slide such as takes place in H. gerbilli, though it suggests
some extra corporeal function, may denote only the escape of trophozoite forms
under the unusual stimulus of removal from the tissues. The conditions relat-
ing to the formation of vermicules in L. canis are in this connection very sig-
nificant. As will be seen later, parasites which invade cells are those in a stage
of development having the chromatin composed of separate rod-like granules,
while parasites which have lain in cells long enough to become encapsuled have
a chromatin mass of quite a different character. Since forms seen invading
cells in the bone marrow never have chromatin like that of the encapsuled forms,
it is clear that these latter do not leave their host cells, in which they have
become encapsuled, to invade others. The behaviour of the encapsuled para-
site, under the stimulus of the conditions in the gut of the tick, is therefore
extremely suggestive of its leaving its capsule normally only in relation to extra
corporeal development.
In the bone marrow, forms are seen resembling those in the peripheral
blood, but there are in addition, other forms which are rarely seen elsewhere,
and others, again, which are confined to this tissue.
Almost as numerous as the encapsuled forms, are parasites entirely different
in appearance. These are round, or egg-shaped bodies, which stain readily and
have no capsule. (Pl. figs. 4 and 5.) They are about the same size, but never
so elongate, as the encapsuled forms, the average measurement being 9 µ across
in the circular forms and 12µ by 8µ in the egg-shaped forms. The protoplasm
in my specimens is most often of the same tint as that of the cell in which it
lies, but it is at times paler or, more rarely, darker. There is usually to be
made out, between the protoplasm of the parasite and that of the cell, a fine
interval. Outside this there is generally a condensation of the protoplasm of
the leucocyte, such as occurs in the encapsuled forms, but this is sometimes
absent or little marked, in which case the presence of the chromatin mass
8
alone attracts one's attention to the parasite.
The chromatin mass is very distinctive, and quite unlike that of the encap-
suled form. It consists of small isolated rod-shaped granules, thirty or forty in
number, arranged loosely in a circle, somewhat as is the pigment in a malarial
crescent. When not free, these forms are invariably found in one kind of cell
only. These cells are of mononuclear type and possess characters which will
be described later.
These round and oval naked forms abound in the bone marrow, but also
occur in much smaller numbers in the liver and spleen ; in the peripheral blood
they are rare. It is undoubtedly this variety of the parasite which James has
noted as being occasionally seen in the peripheral blood. From considerations
that will appear later, it will be seen that he was correct in thinking this to be a
less mature stage of the parasite.
Though, between the encapsuled parasite with reticular chromatin mass
and the naked form with scattered chromatin granules, there is little apparent
connection, yet it is not difficult to find varieties which exhibit every gradation
between the one and the other. Thus some parasites, though they are surrounded
by an oval space, stain readily and exhibit the scattered arrangement of the
chromatin, and others resembling ordinary encapsuled forms so far as their
capsule is concerned, can still be seen to be in a transition stage since their
chromatin consists of a number of aggregated but separate granules. That
one form of parasite changes into the other, and that this change consists in
the development of the naked form into the encapsuled form seems quite
certain. Any doubt of this sequence is dispelled by a study of the changes
which go on in the invaded cell.
I have already briefly touched upon the question of the kind of cell in
which the parasites occur, but since the invasion by a parasite of the leucocytes
whilst these are still in their immature stages in the marrow, is an entirely un-
described condition, it is important, because of the possible bearing of the matter
on the origin of leucocythæmic conditions in man, to ascertain exactly, not
only the behaviour of the parasite, but such cell re-actions as it may give rise
to in its host.
The examination of films of peripheral blood of dogs coloured with Roman-
owski's stains enables certain types of cells to be clearly differentiated.
(a) Cells in which the nucleus is narrow and lobulated, showing a coarse
reticular structure, and the protoplasm of which, when intensely stained,
exhibits numerous fine red granules. Such cells normally form about forty per
cent. of the total number of leucocytes. They are exactly comparable with the
polymorphonuclear leucocytes of man, and resemble these in every particular.
9
In preparations stained with weak Nocht's Romanowski stain, the fine granules
are often not visible, and the protoplasm shows up clear and transparent with a
pinkish tinge, contrasting with that of the second type of cell to be described,
where, under similar conditions, the protoplasm is of a purple colour, owing to
the presence of indistinct granules which have taken up the basic stain. (Fig. 12.)
(b) Cells in which the nucleus may be mononuclear, quadrangular or horse-
shoe-shape, in which the protoplasm when intensely stained exhibits a certain
number of fine red granules together with much bluish granular matter. In
specimens coloured with weak Romanowski's stain the purple or even blue granular
protoplasm contrasts, on the one hand with the clear pink or colourless pro-
toplasm of the polymorphonuclear cells, and on the other with the hyaline blue
protoplasm of those cells which correspond with what, in human blood, one is
accustomed to call large and small mononuclear leucocytes. Though many of
these cells, especially those having horse-shoe-shaped nuclei, approximate in
appearance to the polymorphonuclear cells, they yet form a type so easily
distinguishable as to merit their inclusion under a separate type-head. Such cells
as we have described correspond to the transitional cells of human blood. In the
dog they form about 20 per cent. of the total leucocytes. (Fig. 13.)
(c) Cells of mononuclear type of large size having hyaline basophyllic
protoplasm. Though these cells most resemble the large mononuclear leuco-
cytes of man, they differ from these in possessing more regularly oval nuclei
and protoplasm which stains more intensely. (Fig. 15.)
(d) Cells of intermediate or small size with round nuclei and hyaline some-
what basophyllic protoplasm. They may have comparatively voluminous
protoplasm, or a thin layer only around the nucleus. They often contain a few
large and distinct red granules. Cells of this type are practically identical with
the small mononuclear leucocytes of man. Neither in this nor the last mentioned
type of cell do parasites occur. (Fig, 15.)
(c) Cells with irregular shaped nucleus the protoplasm of which is crowded
with large eosinophyl granules. Though in a few particulars they differ from the
eosinophyl cells of man they undoubtedly correspond to these. They are not as
a rule numerous, but they may constitute as much as 18 per cent. of the total
leucocytes. They are never invaded by parasites. (Fig. 16.)
(e) Mast cells. These resemble the mast cells of man. They are only very
occasionally seen.
In the red marrow, though the great variety of cell forms is at first rather
confusing, it is possible to distinguish with certainty the following:
—
(a ) Cells with large circular or oval nuclei showing fine reticular structure
the protoplasm of which stains more or less markedly blue. These are the
neutrophyl myelocytes. Stained with hæmatin and eosin, their nucleus is pale
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and faintly indicated. I have never been able to detect parasites in this type of
cell. (Fig. 18c.)
(b) Cells with round deeply staining nuclei having a. very coarse reticular
structure, the protoplasm of which is intensely basophyllic (figs. 18 and 19).
These cells are peculiar on account of the depth of stain they exhibit. Next to
the myelocytes and type (c) , they are the most numerous cells in the marrow.
They are not invaded by parasites. (Fig. 18d.)
(c) Cells usually possessing a circular outline and clear or finely reticular
protoplasm. The nucleus is mononuclear, quadrangular, dumb-bell shaped, or
in the form of a horse-shoe. They resemble the transitional cells of the peripheral
blood, though examples of this type with mononuclear nuclei are much more
numerous in the marrow than in the blood. This type is undoubtedly the
polymorphonuclear cell of the blood in its early stages of development. For
convenience of description I shall term this type of cell transitional. It is all
important in the present connection, since it is cells of this type which are
selected by the parasite for its host. (Fig. 18a and 18 b .)
When discussing the relation between the naked and encapsuled form, I
showed that the former was the early stage of the latter. The same relation
holds good in the case of the cell in which these two forms occur. The cells in
which the naked forms occur beyond a slight indentation of the nucleus by the
parasite, exhibit no degenerative changes. They are mononuclear forms of
transitional cells, or more rarely cells of this type having dumb-bell-shaped nuclei.
By the time the parasite has become encapsuled, this cell, which would normally
have progressed through various transitional stages to that of a mature polymor-
phonuclear leucocyte, has by reason of its own development and the action of
the parasite come to possess the fragmented, more or less diffusely staining
nucleus, and transparent protoplasm of the cell we have already described. The
accompanying table, which records in a graphic form parasites encountered in the
examination of the marrow of an infected dog, and the variety of cell they
occupied, shows this sequence very clearly :—
TABLE I.
Circular or oval naked forms with
scattered chromatin.
m
m m m
m m
m m m m m m
Parasites intermediate between encap-
suled and naked forms.
m h m m m m h
Ordinary encapsuled forms .
.
. a
a
a a
a
a
a
a
a
a
a
a
a
a
a
a
a
Parasites encountered are signalled by a letter signifying the type of cell occupied
—
m = M o n o n u c l e a r t r a n s i t i o n a l c e l l .
h = Transitional cell with horse-shoe-shaped nucleus.
a = Altered cell.
f = Free form.
Though in my specimens, the parasite has practically always lain in altered
transitional cells, yet it is quite conceivable that the factor of normal develop-
ment of the cell being greater, or the effect of the parasite less, a condition
would result where the including cell was still recognisable as a polymorphon
u -
clear. This appears to have occurred more frequently in James' than in my
own case, a fact probably due to differences in the age or condition of the dogs
with which we have each had occasion to deal.
It is difficult to believe that the condition of infection described can exist
in any degree without modifying the number and proportion of the leucocytes in
the blood, and since up to the present no parasite attacking immature leucocytes
in the marrow has been known, the nature of any such change could not be
predicted.
Patton has noted in the case of L. funambuli that in severe infections the
number of mononuclear leucocytes is increased, the proportion of these reaching
in some cases 80 per cent., whereas in the normal squirrel (F. palmarum) they
form only about 30 per cent. of the total leucocytes.
James in his memoir gives differential leucocyte counts of four infected
dogs. The number of polymorphonuclear leucocytes in these cases varied
from 58.2 per cent, to 67 per cent., a proportion which does not suggest any
very great increase of their numbers.
In the following table a series of blood counts of healthy and infected dogs
are given. From this it will be seen that the total leucocytes of uninfected dogs
vary between 9,000 and 18,000 per c.m.m. The polymorphonuclear cells form
from 30 per cent. to 64 per cent. and the transitional cells from a 11 per cent, to
26 per cent :—
D o g I . P u p p y
n o t i n f e c t e d .
Dog 2. Puppy
not infected.
D o g 3 . P u p p y
n o t i n f e c t e d .
Do g 4 . P u p p y
not i nf ect ed.
D o g 5 . P u p p y
P r o p l a s m a a b -
unda nt .
D o g 6 . A d u l t n o t i n f e c t e d .
Do g 7 . P u p p y
n o t i n f e c t e d .
D o g 8 . A d u l t P r o p l a s m a
Scant y.
Dog 9. Puppy.
Hæmogr e ga r i
ne s a bunda nt .
Dog 10. Adul t
Hæmogregari nes scanty.
Dog 11. Puppy
Hæmogregari nes abundant..
Dog 12. Puppy
Hæmogregari nes abundant.
Dog 13. Puppy
Hæmogregari nes abundant.
Dog 14. Puppy
Hæmogregari nes abundant.
Polymorphonuclear 59 64 56
53
46 53
56
31 66
42
53
67
61 54
Transitional
. 20 21 24
15
26
15
57
1 1 23
22
15
12
21
1
Large mononuclear 4
6
3
3
6
3
3
4
3
3
4
4
3 1
Small mononuclear 14
9 17
1 1
7 11
16
39
5
25
19
14
11 28
Small mononuclear
w ith granules,
3
20
8
35
Eosinophyl .
.
1 8
2
7
5
4
9
3
4
2
Total leucocytes per
c. m. m.
18,000
9,700
12,700
12,000
6,015 11,400 12,800
23,00
Total red cells per
c. m. m.
4,475,000 1,200,00 0
3,912,500 4,950,000
5,450,000
3,100,000
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