NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
3
of the parasites. A. similar cell (Plate I, fig. 4) was found in a film of finger
blood taken from the second patient two days before his death. This cell con
- t a i n s t h i r t y - t w o p a r a s i t e s s i x o f w h i c h a r e s e e n l y i n g s u p e r i m p o s e d o n t h e
nucleus ; its protoplasm stained dark blue with Romanowsky's stain and was
much vacuolated.
Christophers4 had previously found similar cells in films made from the blood
in large veins, femoral, portal, and hepatic, some hours after death ; and from a
study of sections of the spleen and liver of cases of Kala Azar he concluded
that these cells are derived from the vascular endothelium and that they re-
present the final stages of an endothelial cell which has become distended and
modified by the presence of a large number of parasites. He was able to trace
every gradation of flattened endothelial cell containing only a few parasites to
the enormous cells almost blocking the capillaries in which they lie.
Statham5 w h o h a s s t u d i e d t h e d i s t r i b u t i o n o f t h e L e i s h m a n - D o n o v a n b o d y i n
the tissues confirmed these observations of Christophers and pointed out that
these swollen endothelial cells are met with in the liver, spleen, bone marrow and
lymphatic glands in toxæmic affections and hyperplasias of these organs ; the
researches of Werigo, 6 Gilbert and Carnot 7 a n d o t h e r s h a v e s h o w n t h a t t h e s e
vascular endothelial cells are phagocytic in character and that they take up bac-
teria and malarial pigment.
Marchand and Ledingham,8 who also noted the occurrence of the parasites
in these large cells, consider that only some originate from the vascular endothe-
lium and that the majority are in reality enlarged splenic cells which have taken
on a phagocytic action. It is of interest to note that these large endothelial cells
occur in the peripheral circulation of cases of Kala Azar some days before death,
and it seems most probable that the rupture of these cells and the liberation of
the parasites they contain, explains the occurrence of the large numbers of para-
sites in the peripheral blood of patients suffering from ulceration of the large
intestine. Once the parasites are set free in the plasma they are immediately
taken up by mononuclear, eosinophyl or polymorphonuclear cells.
The presence of these large cells containing numerous parasites in the
circulating blood further suggests the mode of formation of the brawny swellings
which often develop in the later stages of the disease. Captain Long, I.M.S.,
first drew my attention to a patient in his ward who developed such a swelling
on the lower part of the left leg. This patient had many parasites in his
peripheral blood and was at the time suffering from diarrhœa. A painful swell-
ing about 3 inches in circumference involving the skin and subcutaneous tissue
was noticed on the outer side of his left leg. It gradually increased in size
becoming a fluctuating tumour, and was thought to contain pus but, on being
incised, sanious fluid alone exuded. Films of this fluid stained by Romanowsky's
B 2
4
method and carefully examined showed no parasites or bacteria. I have examined
many of these swellings which form in such situations as the face, back. of
the neck, chest, gluteal region, thighs, legs, shoulders or ischio-rectal fossa.
When on the cheek they may result in an abscess opening externally or, as is
more frequently the case, develop into cancrum oris.Another patient under
observation in Captain Long's ward suddenly developed, during the later stages
of the disease, a swelling in the right cheek, which in a few days resulted in a
large sloughing ulcer in the mouth and later spreading outwards to the skin
destroyed all the soft tissues up to the patient's right eye.
The earliest changes towards the formation of cancrum oris appear there-
fore to be identical with the brawny swellings seen in other parts of the body,
and it seems probable that they are produced by a hæmorrhage caused by
the blocking, of a small vessel by one or more of the large cells. It can be
readily understood that the resulting swellings would be liable to be invaded by
bacteria and to develop into ulcers. The sudden appearance of these swellings
also suggests that this explanation of their formation is the probable one.
In the film taken from the patient just before death there were some
parasites showing the earliest changes towards development, and I have on many
occasions seen these appearances in films of peripheral blood. Statham5has
also noticed partial development of the parasites in smears taken from the
organs of a case of Kala Azar 12 hours after death. The parasites measure
from 4µ to 5µ in length, their protoplasm stains a distinct blue with Romanowsky's
stain and often contains three or more vacuoles ; the macronuclei, also enlarged
and stained less deeply; are seen growing towards the centre of the parasites.
Two cells showing these appearances are figured in a large mononuclear
leucocyte in Plate I, fig. 6.
In the same film there was a large body (Plate I, fig. 5) measuring 14 µ in
length containing two parasites. This structure which stained deep blue by
Giemsa's method was of a granular nature and appeared to be similar to bodies
seen in films of splenic blood, in which situation they often contain four or more
parasites. There is considerable difference of opinion as to the true nature of
these bodies ; Laveran, 9 Mesni19 and Donovan10consider they are red blood
corpuscles altered by the presence of the parasite. Christophers11who studied
and described the various appearances assumed by these bodies states they
are detached buds of mononuclear cells or macrophages containing para-
sites. Treutlein1 2
examining some of Rogers' preparations has recently stated
he has seen the parasites in red cells, but no other observer as far as I am
aware has recently described the parasites as occurring in the red corpuscles.
All investigators who have studied the parasites in the human body are agreed
that in sections of the organs they are never seen in the red cells.
5
The Development of the Parasite in the Bed-Bug.
On December 27th, twelve bugs were fed between 9 and 10 P.M. on the
patient mentioned above, and on the 31st one male and three adult females were dis-
sected between 8 A.M. and 12 noon, the midguts and their contents being smeared
out on slides and stained by Giemsa's method. In these films parasites were
found in an unchanged condition (Plate I, fig. 7 a) and in all stages of develop-
ment up to the mature flagellates. The leucocytes were seen as indistinct
granular masses, with or without a central deeper stained area, the remains of the
nucleus ; such disintegrating cells may sometimes be seen in the midgut as late
as the fourth day, but as a rule they disappear by the end of the third.
The parasites are ingested by the bug while lying in the polymorphonuclear,
eosinphyl, large mononuclear, and endothelial cells, and from a number of
earlier observations it appears that the majority remain unchanged until the
second day. While still lying in the disintegrating leucocytes they begin to
show the earliest signs of development ; the protoplasm increases in volume,
is more granular and instead of staining a faint pink with Giemsa's stain, now
appears blue throughout and contains either one large central vacuole (Plate I
fig.7 b) or a number of smaller ones (Plate I, figs. 7 d, e,f ).
Coincident with these changes the macronucleus becomes enlarged and
stains less deeply ; its central dark chromosome divides into a number of rods
which are distributed throughout its substance. It may now be either circular
in shape or oval with one end more pointed, and may lie at the periphery or
across the centre of the parasite. The micronucleus, still rod like and staining
almost black, increases in size and is usually seen lying towards the periphery
of the cell. Parasites more advanced towards development have a very charac-
teristic appearance ; their protoplasm has still further increased in amount so
that the body of the parasite is made up of granular blue staining protoplasm.
The vacuoles also appear more distinct measuring from .8µ to 1µ in diameter ;
they are usually circular in shape and vary in number from four to eight. When
deeply stained by Giemsa's method the periphery of these parasites which stains
a much darker blue than the rest of the cell suggests a condensed layer of proto-
plasm of the nature of a cuticle. Parasites exhibiting these changes vary in
size from 4µ to 7µ ; some are oval in shape but the majority are round. In a few
the macronuclei have still further increased in size and show the earliest changes
towards division (Plate I, fig. 7 f ). A constriction forms on the inner side and
gradually deepens ; at the same time the chromosomes begin to pass to the two
ends which become rounded off, and on a similar constriction forming on the
opposite side the appearance of a dumb-bell is produced (Plate I, fig. 7 c and
fig. 8 f ), and later two daughter nuclei are formed (Plate I, fig. 9 f ).
6
Plate I, fig. 7 c , shows five parasites which have been lying in one leucocyte,
and which, as this has degenerated, have become grouped together ; one of the
parasites is still in an early stage of development while the others are much
further advanced. Vacuolation at this stage and later is a marked feature of the
development in the bug, many of the parasites containing four or five vacuole
in close apposition but separated by strands of dark protoplasm.
From a careful study of the films it was seen that after the early stages
described above one or other of two things takes place : (1) the parasites may
rapidly pass on to flagellation ; or (2) what is more important, they further enlarge
and by consecutive division of the macro-and micronuclei produce a rosette of
from four to eight parasites. Judging from the large number of these rosettes.
and groups of elongated flagellates still attached, it appears that the further
increase in size and resulting formation of rosettes is the more usual change that
takes place.
In the enlarged parasites an area staining bright pink and with a darker
cent r e meas ur i ng about . 4µ devel ops i n cl os e r el at i on t o t he mi cr onucl eus ( Pl at e
I, fig. 8 a, b, c, d, e,f ). This structure which represents the early formation of
what has been variously named ' vacuole-like area' (Christophers 1 3
) , ' F l a g e l l a r
vacuole' (Leishman14), and 'Eosin body' (Rogers1 5
) r a p i d l y i n c r e a s e s i n s i z e a n d
when fully developed is almost circular in shape measuring from 1µ to 3µ,
according to the size of the parasite (Plate I, fig. 9 d). When stained deeply
with Giemsa's stain it is seen to consist of a homogenous pink substance in
which darker pink strands are scattered irregularly. It now passes to the surface
of the parasite when it ruptures and a small pink brush begins to protrude
directly from it (Plate I, fig. 9 a
and
b). When stained deeply and examined
with a high magnification this brush is seen to consist of a number of delicate
pink strands lying in a pink matrix and in suitable preparations the filaments
can be traced into the vacuole to one or two dark pink dots in the
n e i g h b o u r h o o d o f t h e m i c r o n u c l e u s ; b u t i n n o n e o f t h e s p e c i m e n s w a s i t d i r e c t l y a t t a c h e d
to this structure (Plate I, fig. 9 b, c, e and f ). The protrusion of the brush
of filaments progresses rapidly and is seen in all stages from a relatively short
curved rod (Plate I, fig. 9 c, e and f ) to the long fully developed wavy flagellum,
measuring from 15µ to 20µ (Plate II, fig. 1a ) .
From a careful study of the formation of the flagellum I believe that it
forms within a vacuole and that the pink area seen when parasites are stained by
Giemsa's method is due to the deposition of colouring matter in the vacuole.
The fact that after the extrusion of the flagellum the vacuole collapses and
then appears as a pink band on either side of the insertion of the flagellum
further suggests its relation to the flagellum, and I therefore propose adopting
t h e n a me ' f l a g e l l a r v a c u o l e ' g i v e n i t b y Le i s h ma n .
7
Many of the round or oval parasites are seen in all the stages of flagellation
while lying side by side and in some the division of the macronucleus has
already taken place (Plate I, fig. 9f ). In some of these parasites the flagellum
was seen to end in an achromatic zone close to the micronucleus and Plate II,
fig. 1a , s h o w s a p a r a s i t e i n w h i c h t h e f l a g e l l u m p a s s e d a l i t t l e b e y o n d t h e
micronucleus to end in a pink dot.
MacNeal16 who has studied the attachment of the flagellum of
T r y p a - n o s o m a l e w i s i
s t a t e s t h a t i n f a v o u r a b l e s p e c i m e n s i t c a n b e t r a c e d t o a n
achromatic space in close proximity to the micronucleus ; Laveran and Mesnil1
have also noted this appearance in the same trypanosome.
Considerable variation is met with in the further stages of the first type
of development described. In many parasites, as has been mentioned above
before flagellation has begun, the macronucleus divides into two, and this is
followed by the enlargement and division of the micronucleus. Two flagellar
vacuoles develop close to the micronuclei and in some cases are seen fused
together ; the two flagella rapidly pass out of them and as division proceeds
the flagellates are seen attached by their flagella alone (Plate II, fig. 1 c ) o r
by both poles. In other cases the parasites increase in size and become much
vacuolated, the macronucleus enlarges and shows signs of division while the
micronucleus has already divided. Two flagella now develop a little distance
apart and later become extruded and on completion of the division of the macro-
nucleus the parasite splits longitudinally into two spindle-shaped cells (Plate II,
fig. 1 b ). Some of the enlarged oval flagellates also showed signs of elongating
into these spindle-shaped forms (Plate II, fig. 1 a a n d
c ) .
It will thus be seen that a certain percentage of the developing parasites
pass on to flagellation and then divide longitudinally to form oval or
s p i n d l e - s h a p e d f l a g e l l a t e s ; t h e r e b e i n g n o r e g u l a r i t y o b s e r v e d i n t h i s p r o c e s s .
In the second type, development which results in the formation of rosettes
consisting of from four to eight flagellates also follows on the early enlargement
of the parasites. The macro-and micronuclei divide and pass to the opposite
sides of the cell (Plate I, fig. 10), which has a globular appearance and is rich in
granular protoplasm. One of these macronuclei divides again while the other may
as yet remain unchanged or only show ? an increase in size ; but the micronuclei
during this time divide rapidly so that while there may be only two macronuclei,
there may be three or more micronuclei (Plate I, fig. 10 e). Fig. 10 d and f
shows the method of division of the micronucleus ; after it has become
thickened and considerably elongated it divides transversely into two, a method of
division which is maintained throughout all the later stages of the development.
The division of the macro-and micronuclei and the further growth of the
cell result in the formation of a body measuring from 10µ to 12µ in diameter ;
its protoplasm stains reddish blue with Giemsa's stain and contains groups of
vacuoles (Plate I, fig. 10). Such a cell may contain from four to eight macro-
and micronuclei grouped about its centre. The flagellar vacuoles now develop
close to the micronuclei and later all the flagella pass out from one side of the
cell (Plate II, fig. 1d a n d
e ) . S e p a r a t i o n o f t h e f l a g e l l a t e s n o w b e g i n s a t t h e
flagellar end and transparent lines pass up through the cell, and from four to eight
fully developed flagellates become separated and swim away.
These flagellates vary from 6µ to 12µ in length and from 4µ to 5µ in
breadth, their size depending on the rapidity with which the rosette breaks up;
the majority are elongated and it is from further growth of these that the long
free swimming forms are produced. It is important to note that equal longi--
tudinal division of these flagellates while still attached or when free was not
observed.
Plate II, fig. 3 c, shows a rosette in the process of division; the micronuclei
are seen in the two parts undergoing further division, while distributed along
the sides and periphery of the cell there are eight micronuclei and six flagella.
Such a cell, it could be readily understood, might produce even more than eight
flagellates. Plate II, fig. 3 b , also shows a small rosette of four elongated flagel-
lates and two cells which have become separated from a rosette containing
six parasites.
The oval or spindle-shaped parasites which are formed either from the
round flagellates or from their further division exhibit very characteristic appear-
ances. W hen stained deeply by Giemsa's stain they contain dark blue granular
protoplasm and a number of vacuoles of varying size; their macronuclei, usually
circular in shape and situated about the centre, stain deeply pink and contain a
number of dark chromatic rods. The micronuclei generally lie across the long
diameter of the cells near the macronuclei, seldom at the anterior ends (Plate II,
fig. 2). This position of the micronuclei, as I shall mention later, indicates
that the cells are about to undergo further division. The flagellum, which stains
deep pink, consists of a number of filaments adhering closely to each other and
they are inserted in a pale area close to the micronucleus; in none of the
specimens was the flagellum seen attached along the side of the cell.
In these oval and spindle-shaped parasites further changes soon begin to
take place; the macronucleus enlarges and spreading to the sides of the parasite
occupies the whole of the centre of the cell, and its chromosomes become
distributed throughout its substance. The micronucleus at the same time
enlarges and later elongates, changes which are seen preparatory to division.
The flagellum thickens and commences to divide. If this is relatively short
the split extends throughout its length; but if it is long the basal portion
alone thickens and the division then extends through about a quarter of its
9
length, the new flagellum beginning to separate first at its anterior end (Plate II,
fig. 2 a). In parasites showing a later stage of division the two flagella are
completely separated and end close to the micronucleus. Preparatory to division
the macronucleus in many of the cells shows a dumb-bell appearance and the
two portions of the micronucleus which has divided transversely are seen side by
side. Eventually a transparent line developes through the centre of the parasite,
the micronuclei separate further, the macronucleus divides, and on the line
of separation passing through the parasite two oval cells are produced (Plate II,
fig. 2). If a spindle-shaped flagellate is an unusually large one, its division
generally results in two smaller spindle-shaped cells, though it may produce
two elongated parasites which are either seen still attached by their anterior ends
or by both poles (Plate II, fig. 2 c ). In some of the spindle-shaped cells a
process of unequal longitudinal division was observed, a thinner parasite
splitting off as described above (Plate II, fig. 2 b).
The majority of long flagellates shown in Plate II, fig. 4, are formed from
the rosettes, a smaller number, however, particularly the thinner ones, may
result from the division of the spindle-shaped cells. These long flagellates vary
from 12µ to 20µ in length and from 4µ to 5µ in breadth, their macronuclei are
oval in shape, stain dark pink and contain a number of rod shaped chromosomes.
The anterior poles of the cells are rounded, their bodies exhibit undulations and
end posteriorly in pointed beaks; their protoplasm stains dark blue containing a
number of lighter areas but no vacuoles or chromatic granules. The flagella are
thick filaments measuring from 16µ to 24µ in length ; they originate in one or
two chromatic dots usually close to the micronuclei and pass out of the cells
through the centre of the anterior ends. The micronuclei are always seen lying
transversely to the long axis of the parasite about 1.5µ to 2µ from the anterior
extremity. In many of the parasites the remains of the flagellar vacuoles are
seen as indistinct pink areas surrounding the insertion of the flagella ; in others
they are represented by a pink line on either side of them. None of these fla-
gellates were seen showing equal longitudinal division, and it appears doubtful
whether this method of fission does take place in these cells.
The above were the only changes observed in the four films under discussion,
and it will be seen that after lying 80 hours in the midgut of the bug some of the
parasites were still unchanged, though the majority had passed through all the
stages of development up to flagellation.
On January 1st two male and two female bugs were dissected between 12
noon and 1.3o P.M., contents of their midguts being smeared out on slides and
stained by Giemsa's stain ; the films from one female bug contained many
parasites, but the others very few.
In each slide there were one or more parasites in an unchanged condition,