NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
10
as well as some showing the earlier changes towards development, and in the film
made from the female bug there were also a number of rosettes and long free
swimming flagellates. In addition to the above this film contained a number of
pairs of small attenuated flagellates measuring from 6µ to 8µ in length and 2µ
to 3µ in breadth. Their protoplasm stained dark blue but contained no vacuoles
or granules ; the macronuclei were circular or oval in shape occupying nearly the
whole of the centre of the parasite. In some of the cells the macronuclei
showed evidences of further division, having a bilobed appearance, each lobe
containing a number of chromatic rods (Plate II, fig. 5 a) . The micronuclei in
the majority of the cells lay close up to the macronuclei and the flagella were
comparatively short, measuring from 8µ to 12µ in length.
These bodies were attached to one another either by their posterior poles or
along the whole length of the body, and it is evident that they had originated
from the further longitudinal division of the oval parasites which result from the
division of the spindle-shaped ones. I was unable to satisfy myself as to
whether these small parasites may have been produced from the long flagellates
seen in Plate II, fig. 4, as the majority of these forms showed no evidences of
longitudinal division.
Plate II, fig. 5 b , shows a large oval flagellate measuring 8.5µ in diameter
with a smaller parasite splitting off from its side. The macronucleus of the
parent cell occupies its posterior pole and shows signs of division. The free
portion of the flagellum of the large cell is considerably thickened almost to its
end, while the attached part is divided into two filaments which pass into a large
flagellar vacuole just anterior to the micronucleus. The macronucleus of the
smaller cell is also situated at the posterior pole and its micronucleus lies close up
to it. Its flagellum is almost separated from that of the parent cell being
attached by its end alone. The commencing division of the flagellum and macro-
nucleus of the parent cell and the position of its micronucleus show that it is
about to divide longitudinally.
On January 2nd the remaining bugs, three adult males and one female, were
dissected between 12 noon and 1-30 P.M., the midguts being dissected out and
prepared as described above ; in addition smears were made from the crops and
salivary glands of all the bugs. In two films, one prepared from the midgut of a
male and the other from that of a female, there were a number of parasites, some
exhibiting the earliest changes towards development, others more advanced, as
well as a few rosettes and small flagellates.
Plate II, fig. 5 e and f, shows two parasites in the early stages of develop-
ment ; in the smaller the flagellar vacuole is seen as a small pink area lying near
the micronucleus. The larger parasite measures 8µ in length and 4.5µ in
breadth, one end being rounded and the other pointed ; its protoplasm stained
dark blue with Giemsa's stain and contained some small pale areas, but none of -
the distinct vacuoles usually seen at this stage. Its micronucleus a little
enlarged lay close to the macronucleus which is round and compact staining deep.
red.
Plate 11, fig. 6 a, shows another cell of a similar nature, though it is almost.
round its protoplasm is less deeply stained and contains a few indistinct vacuoles.
Its macronucleus lying near one end is almost divided into two, while the micro-
nucleus is situated about the centre and lying near it is a faint pink mass.
suggesting the early development of a flagellum. These parasites clearly show
that the Leishman-Donovan body may remain in the midgut of the bug for at
least five days before they begin to develop.
The rosettes seen in these slides are similar to those described above, the
majority consisting of fully developed flagellates. Plate 11, fig. 5 c, shows a long
flagellate which measures 9 . 5µ in length and 2 . 5µ in breadth; its oval.
macronucleus is situated about the centre of its body and lying close to it and.
about 1.5µ from the anterior end is the micronucleus. The posterior end of the
parasite is pointed and situated about 3µ from this end there are two small pink
masses which on higher magnification are seen to consist of a number of chro-
matic granules. This is the first parasite that has shown this appearance,
though in another long flagellate (Plate 11, fig. 4) there was a suggestion of the
same structure. From these isolated specimens it is impossible to say what this.
represents, but further reference will be made to it later.
Plate 11, fig. 5 d, represents two attenuated flagellates attached by their
poles, and from the outer side of one of the cells a spirilla-like parasite is seen.
splitting off. The macronuclei of the two parasites are circular and compact.
staining uniformally dark red or almost black with Giemsa's stain ; in one cell
the macronucleus is near the anterior end and in the other towards the posterior
pole, and in each case the micronuclei are lying close up to their respective
macronuclei. The protoplasm of these cells stains dark throughout and is
devoid of granules or vacuoles. The spirilla-like parasite is 5.4µ in length and
1µ in breadth, and in its protoplasm there are two small chromatic granules, the
anterior being distinctly rod shaped.
These pairs of attenuated flagellates are seen in all stages of longitudinal
division ; some are without flagella and are similar to those described and figured
in my first report which therefore represent a very late stage in the development
of the parasite. Plate 11, fig. 6 c, represents one of these attenuated flagellates
lying free ; it measures 9µ in length and 1. 2µ in breadth, and its flagellum
measures 10µ. The macronucleus, which consists of a number of chromatic rods,
is situated about the centre, while the micronucleus is 2µ from the anterior end.
The protoplasm of the cell appears very granular and stains deep blue with
C 2
12
Giemsa's stain. The flagellum, a stout filament, is inserted into the body near
the micronucleus, though not attached to it.
Another type of small flagellate dividing longitudinally is shown in Plate II,
fig. 6 b. Its two daughter cells further show evidences of multiple division of
their macro-and micronuclei. In one of these there are two macronuclei, one
circular in shape situated at the posterior pole, the other having a dumb-bell
appearance situated about the centre. There are also three micronuclei in this
cell, two near the central macronucleus and the other towards the posterior end.
In the second cell there is a single macronucleus lying about the centre with the
micronucleus near it. Two flagella are seen passing out from one cell and one
from the other. Fig. 6 d shows two flagellates in each of which there are two
macronuclei, two micronuclei and a single flagellum.
The process of irregular division in the larger oval forms undoubtedly
results in the formation of small irregularly shaped flagellates shown in Plate II,
fig. 6 e and f ; one of these small cells shows a dumb bell shaped macronucleus
and three micronuclei. Its posterior end is distinctly pointed while the rest of
the cell is rounded. Fig. 6 f represents two small flagellates attached by their
posterior poles in which the round macronuclei are lying with the two micro-
nuclei near them. The protoplasm of all these small cells stains uniformly
dark blue with Giemsa's stains and contains heither vacuoles nor granules.
Plate II, fig. 6 g, shows a large flagellate measuring 15µ in length; two
macronuclei are seen lying on each side of the centre of its body and a little
posterior to one there is a single chromatic dot. One micronucleus is 2µ from
the anterior end and the other about 5µ, and the flagellum is inserted near the
anterior one. Two chromatic filaments are seen extending from the vicinity of
the macronuclei, and uniting lower down pass into the flagellum. This was the
only flagellate exhibiting these appearances, and later when discussing the
development of the parasite reference will again be made to it. I was unable to
find any parasites in the smears made from the crops and salivary glands of the
bugs.
There can be no doubt from the above description that the Leishtman-
Donovan bodies had undergone marked developmental changes in the bed-bug
(Cimex rotundatus), the following being a summary of the nature and order of
sequence of these changes. On the second day after being ingested by the bug
the parasites begin to develop, their protoplasm increasing in volume ; at the
same time the macronuclei enlarge and show signs of commencing division.
The parasites may now either pass on to flagellation, or further growth followed
by the consecutive division of the macro-and micronuclei may result in the
formation of rosettes. The single flagellates enlarge and begin to divide longi-
tudinally to result in oval or spindle-shaped cells ; while the rosettes after
13
flagellation begin to divide up into separate elongated flagellates. The oval
or spindle-shaped parasites divide repeatedly either by equal or unequal longi-
tudinal division and result in smaller and more irregular forms. All these
changes may be seen in the bug during the first three days. Still later longi-
tudinal division of the oval and irregular flagellates progresses rapidly, so that by
the fifth day the majority have become small or spirilla-like flagellates.
As the bugs were dissected at intervals of about 24 hours it was difficult
to follow some of the later stages of the development. For instance, it is
:probable many spindle-shaped or oval cells are produced directly from the
rosettes, and further the long fully developed flagellates may be formed from
these spindle-shaped cells. I was also unable to find any evidence to show
that the long flagellates may divide longitudinally, and none of them had the
Spirilla-like forms splitting off as described by Leishman.
The small irregular flagellates containing more than one macro-and micro-
nucleus figured in Plate II, fig. 6, result most probably from the rapid and
irregular division of the oval forms produced from the spindle-shaped parasites
Further, it must be remembered that the bugs had not fed for five days, so that
it is quite possible some of the later changes were considerably modified by the
absence of blood.
During the six days the parasites were developing the bugs were kept in an
open verandah where the average temperature was 72°F. From the fact that
in cultures the parasites only develop below a temperature of 75°F, Rogers13
believes that the majority of patients become infected with the Leishman-
Donovan body during the cold season. He finds from an analysis of two series
of cases in Calcutta, that the disease commenced during the cold or early in
the hot weather. Dr. Dodds Price18 also states that in his European cases and
in the majority of his native patients the disease began during the cold weather.
From these observations it appears that the disease usually begins in the cold
season, and if, as Rogers states, the incubation period is probably a long one, it is
natural to conclude that the patients were infected months earlier in the hot season.
As stated in my first report2 I found that the parasites developed into
flagellates when the temperature was between 8o° and 82° F. ; and as we do not
know even the approximate length of the incubation period, it seems premature
to conclude that the infection can take place only during the cold season. A
large number of experiments with infected bugs kept at different temperatures
are necessary before we can hope to solve this point.
On comparing the development of the parasite in the bug with that seen in
cultures in vitro as described by Rogers,15 Christophers,13 Leishman and
Statham, 14 it will be seen that though they agree in many points, they differ
considerably in others. Vacuolation of the protoplasm in the earlier stages of
14
development is seen in both the bug and in the cultures, but it was absent in the
later states in the development in the bug. The chromatic dots which Leish-
man has described occuring in pairs in the protoplasm both in the younger stages
prior to exflagellation as well as in the mature parasites were not seen in any of
my films. Christophers and Leishman have described the formation of two or
more parasites resulting from the consecutive division of the macro-and micro-
nucleus of a single cell, and the latter mentions that these cells may give rise
to three or even four individuals by a process of further sub-division of the
macro-and micronuclei. This process is apparently a marked feature of the
development in the bug resulting in the formation of rosettes of from four to eight
flagellates.
In the bug longitudinal division of the round or oval parasites was not seen
prior to flagellation, though in some of the parasites the macronuclei showed
evidences of division; all the twin parasites had grown to maturity while lying
side by side. This method of multiplication is therefore entirely limited to the
flagellate stage.
In the majority of the spindle-shaped and oval flagellates the micronuclei
were seen close up to the macronuclei, seldom at the anterior end; in the long
mature flagellates and spirilla-like forms, however, the micronuclei were situated
some distance from the macronuclei and about 1 . 5µ to 2µ from the anterior
ends. In the case of Trypanosoma lewisi when a parasite is about to divide
the blepharoplast passes close up to the nucleus, only resuming its final position
when the cell assumes its adult form. When this position of the micronucleus
is seen in a young flagellate of the Leishman-Donovan body, it indicates that
the cell has not yet reached maturity and that further division is about to take
place. In my first report I drew attention to a few instances of parasites
showing this appearance, but I was not then able to explain its significance.
A number of attenuated parasites were seen free, and in one case a thin cell
containing two chromatic dots was still attached to a small flagellate. Multiple
division of the macro-and micronuclei of the small oval flagellates takes place in
the bug about the fifth day resulting in the formation of still smaller flagellates
which are themselves capable of further longitudinal fission. So far as I am aware,
this process has not been seen in cultures. In the later stages of the develop-
ment of the parasite in the bug two distinct types of flagellates were seen; a
long relatively thick form measuring from 9µ to 15µ in length and 2µ to 3µ in
breadth, and a small irregular flagellate measuring from 4µ to 6µ in length and
1µ to 4µ in breadth. In both the micronuclei are situated about 2µ from the
anterior ends through the centre of which the flagella pass out.
There was no evidence to show that the thin parasites originated from the
larger ones, though Leishman has observed this process in cultures, and further,
in the median line the thick first segment of the labrum which is continuous with
the dorsal integument of the head. The antennæ consist of four segments, the
first two being the thicker, while the third and fourth are slender and covered
with long hairs.
The prothorax is semilunar in shape with two rounded horns extending
close up to the eyes; its upper surface is raised in the centre and towards the
sides, ending abruptly at a line a little beyond the level of the eyes; the re-
mainder of the surface including the two horns is flattened from above down-
wards. The ventral surface of the prothorax is concave and hollowed out on
each side of the mid-line where the first pair of legs are inserted. The
mesothorax as seen from the dorsal surface is triangular in shape with its apex
projecting over the metathorax and between the elytra. The metathorax next
in size to the prothorax is almost entirely covered on its dorsal side by the elytra,
and on the ventral surface is seen as a small cleft on the inner side of the
middle coxa. The elytra which are inserted into the mesothorax just below the
lateral angles are two rudimentary scallop-shaped pieces of chitin lying over
the metathorax and sides of the first abdominal segment. Their dorsal surfaces
are convex and covered with bristles, while their ventral surfaces are concave.
The abdomen is rounded and consists of seven segments with an eighth
anal appendage. It is broadest at the third segment and gradually becomes
narrower towards the end where it is covered with long hairs. In the
male the penis is seen flexed in a notch between the seventh and eighth
segments.
This bug is distributed throughout Europe and North America, and is also
found in Suez, Egypt, the Sudan, the North-West Frontier Province of India,
China, South Africa and Australia.
Cimex rotundatus Sign or the Indian bed-bug is darker than the above
species being of deep mahogany colour; its head is not as long or as broad as that
of lectularius. Its prothorax is also narrower and shorter, is more rounded and
not flattened at the sides as is the prothorax of the type Species. Its abdomen
is less orbicular, being broadest at the second segment and tapers more abruptly
towards the end; in all other respects it is similar to Cimex lectularius.
Cimex rotundatus is distributed throughout India, Burma, Assam, Malay,
and is also found in Aden, Sierra Leone, the Islands of Mauritius, Reunion,
St. Vincent and Porto Rico.
Cimex pipistrelliJ e n y n s i s s i m i l a r i n c o l o u r t or
o t u n d a t u s ,
its prothorax is.
also less marginal and in all other respects it is more closely allied to the Indian
bed-bug.
Cimex columbarius Jenyns has a similar prothorax to lectularius and is
also of the same colour.
Dissection of the bed-bug. The following method of dissecting the bed-
bug has been found satisfactory. After having killed the insect by placing it in
a tube with a plug of cotton wool containing a few drops of chloroform, it is
taken up with a fine pair of forceps and the legs are pulled off. In removing
the fore legs it is necessary to be careful not to injure the prothorax, as they are
firmly fixed in concavities on its under surface. The elytra are next removed
by raising them with a fine pair of forceps and gently twisting them off from
their joints at the angles of the mesothorax. The bug is now placed in a drop
of normal saline solution with its head directed towards the dissector. A fine
needle is inserted with the left hand into the right side of the prothorax, while
with another fine needle in the right hand the joint between the prothorax and
mesothorax is separated. By gently drawing the needle in the left hand and by
exerting pressure on the dorsal surface of the abdomen with the other
needle, the œsophagus, midgut and the remaining part of the intestinal tract
are drawn out, and any portion can then be isolated and examined. The salivary
glands lying on each side of the œsophagus are also exposed by this method.
The ducts of the salivary glands and the œsophagus can be followed into
the crop situated in the head by carefully removing the prothorax.
Conclusions.
1. Though Kala Azar is a chronic disease lasting many months and often
years, it occasionally runs an acute course terminating in from four to five months
as illustrated by the case described above. In these acute cases as well as in
the chronic ones terminating with ulceration of the large intestine, the parasites
are found a few days before death in large numbers in the peripheral blood in
the leucocytes and endothelial cells, and the latter are probably the source of all
the parasites seen in the circulating blood.
2. Though the parasites are most abundant in the peripheral blood towards
the end of the disease, they are also found in the early stages as a case recently
seen clearly illustrated.
3. In the female as well as in the male bed-bug (Cimex rotundatus) the
parasites have by the third day passed through all the intermediate stages of
development described above up to the formation of the mature flagellates.
Rapid multiplication by rosette formation is a characteristic feature of the deve-
lopment of the parasite in the bed-bug. As the male bug sucks blood it probably
plays as important a rôle in the transmission of the disease as the female bug.
4. The infection acquired by the bug varies considerably, some ingesting
large numbers of parasites, others only a few; and there is no evidence at present
to show that the development in the bug depends on variations in the tempera
ture.
5. The tendency that the disease has to linger in a house for a long time is
probably explained by the fact that the parasite may remain in the midgut of
the bug for several days before beginning to develop, and, as the nymphs which
take from seven to ten weeks to arrive at maturity, may ingest the parasites
shortly after hatching, and as a rule feed only once between each moult, the in-
fection may remain for a considerable time in a house ; there is no evidence at
present to support the view that the infection is inherited by the bug.
I wish to take this opportunity of thanking Mr. Lounsbury for specimens of
Cimex lectularius L. from South Africa, Mr. W. Rainbow, Australian Museum,
for specimens of Cimex lectularius from Sydney, Dr. C. W. Branch for a large
collection of Cimex rotundatus Sign. from the Island of St. Vincent, Dr. J. W.
W. Stephens for specimens of Cimex rotundatus from Sierra Leone, Dr. C. A.
Butler, United States Naval Hospital, San Juan, for specimens of , Cimex
rotundatus, and Mr. W. L. Distant for information and advice.
REFERENCES TO LITERATURE.
1 .
P a t t o n . — P r e l i m i n a r y R e p o r t o n t h e d e v e l o p m e n t o f t h e L e i s h m a n - D o n o v a n b o d y
in the bed-bug. Scientific Memoirs by Officers of the Medical and Sanitary
Departments of the Government of India. New Series, No. 27.
2. Signoret.—Notice sur quelques hemiptera nouveaux. Ann. Soc. entomol., France,
1852, X, p. 539.
3. Patton.—Notes on the distribution of the two species of bed-bug. Indian Medical
Gazette, Vol. XLII, No. 2, February 1907.
4. Christophers.—On a parasite found in persons suffering from enlargement of the
spleen in India. Scientific Memoirs by Officers of the Medical and Sanitary
Departments of the Government of India. New Series, No. 11.
5. Statham.—A case of Kala Azar, Journal of the Royal Army Medical Corps, Vol.
V, September 1905.
6. Werigo.—Annales de l'Institut Pasteur, 1892.
7. Gilbert and Carnot.—Les fonctions hepatiques. Paris.
.8. Marchand and Ledingham.—On the question of Trypanosoma infection in man.
Lancet, January 16th, 1904 ; Centralblatt far Bakteriologie, Originale,
B. XXXV, No. 5, 1904 ; Über infektion mit Leishman' schen Korperchen und
ihr. Verhaltnis zur Trypanosoman-Krankheit. Zeitschr. f. Hygiene und
Infektions Krankeiten, B. XLVII.
.9. Laveran and Mesnil.—Suru n P r o t o z o a r e n o u v e a u P i r o p l a s m a D o n o v a n i ( L ay
. e t .
Mesn.) parasite d'une fiévre de l'Inde. Comptes Rendus des seances de
l'Academie des Secinces. Tome CXXXVII, p. 957, Seance du 7 December
1903.
1 0 .D
o n o v a n . — H u m a n P i r o p l a s m o s i s . T h e L a n c e t , S e p t e m b e r l o t h , 1 9 0 4 , a n d
January 2 1st, 1905. Human Piroplasmosis. British Medical Journal, Sep-
tember 17th, 1904.
1 1 .C
h r i s t o p h e r s . — A
p r e l i m i n a r y r e p o r t o n a p a r a s i t e f o u n d i n p e r s o n s s u f f e r i n g
from enlargement of the spleen in India. Scientific Memoirs by Officers
of the Medical and Sanitary Departments of the Government of India.
New Series, No. 8.
12. Treutlein.—Uber Protozcenblutkrankheiten bei Mensch und Tier in Indien und
Deutsch Ostafrika. Munch Med. Wochenschrift, Mai 1st, 1906.
73. Christophers.—On a parasite found in persons suffering from enlargement of the
spleen in India. Scientific Memoirs by Officers of the Medical and Sanitary
Departments of the Government of India. New Series, No. 15.
74. Leishman.—The development of the Leishman body in cultivation. Journal of
the Royal Army Medical Corps, Vol. IV, March 1905.
Rogers.—Further work on the development of the Herpetomonas of Kala Azar
and Cachexial fever from Leishman-Donovan bodies. Proceedings of the
Royal Society. B. Vol. 77, 1906.