NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
13
parasite ; and moreover the method of spread of Kala Azar in Madras is strong-
ly opposed to the view that the parasite is carried by a flying insect.
Further, there is no proof that Franchini has actually observed the post-
flagellate stage of the parasite, for he makes no mention of the possibility of his
mosquitoes being infected with a herpetomonas. In Madras, Neocellia stephensi
is infected with a species of herpetomonas which is transmitted through the
larva, and it is very similar to the parasite of Kala Azar. Franchini's mos-
quitoes were apparently caught at large, and not bred from the egg, which is the
only possible way of excluding a natural herpetomonas. An important point to
note in connection with this flagellate of anopheles mosquitoes is that it may
pass back to its post-flagellate stage in the midgut of the insect. Recently
when in Bombay, Dr. Bentley kindly showed me in the fresh condition, the
midgut of an anopheles which was packed with the post-flagellate forms of this
herpetomonas and I have long known that these mosquitoes may pass out the
flagellate stages in their excreta. From my observations on the life history of
the parasite of Indian Kala Azar in Cimex rotundatus and Cimex lectularius,
I find it difficult to understand how the allied parasite undergoes the final
stages of its development so rapidly in. anopheles, and how it can again
pass back to man through the excreta of mosquitoes.
I will now shortly mention some points in connection with Basile's trans-
mission experiments with fleas. In the first place it is necessary to note that
canine Kala Azar is widespread in Italy, and as it is a comparatively benign
disease, it is difficult to exclude it in such experiments. Secondly, I note that
Basile makes no mention of the possible confusion between the parasite of
canine Kala Azar and Piroplasma canis in the case of his dogs. I do not in
any way wish to throw doubt on the accuracy of his observations, but they
would have been much more convincing had this common piroplasm of dogs
been mentioned. It is however by no means a remote possibility, for I have
repeatedly seen P. canis in the spleens of dogs, morphologically indistinguish-
able from the parasite of Kala Azar. When inoculating dogs with the Indian
parasite, this possibility was foreseen and every precaution was taken to exclude
it, not only by examining the blood of the dogs for a considerable time, but by
removing all ticks. In this way I failed to obtain any positive results, though
one of the dogs was kept for many months.
The Life History of the Parasite in Cimex rotunclatus and Cimex
lectularius.
For several months before the experiments I am about to record were
begun, adult bugs both Cimex rotundatus and Cimex lectularius were collected,
and fed in the laboratory. Their eggs were separated, and the first nymphal
instars of each species were obtained. The adults of Cimex rotundatus were
collected in Saidapet, while those of Cimex lectularius were imported from
London ; I wish to take this opportunity of thanking Miss Blakeney of the
Mildmay Mission Hospital for the great trouble she took in obtaining this species
for me. As before, the bugs were kept on pieces of sterilised filter paper in
glass tubes, and were thus readily fed on any part of the body.
On January 26th I was informed that there was a suitable case in the
General Hospital, and on visiting the patient I examined a film of finger blood and
found. in it 23 parasites. The following is a short history of the case :—Mr. R.,
aged 40, a Eurasian of independent means, had been suffering for the last six
months with low fever, and he noticed that he was slowly getting thinner.
About the end of December 1911, he developed an attack of diarrhœa, with great
pain in the region of his large intestine ; he gradually became worse and the
diarrhœa now was more of a dysenteric nature, blood and mucus being fre-
.quently passed with much tenesmus. As he began to get rapidly worse, and was
having about thirty motions in the twenty-four hours, he sought admission in
the General Hospital. On examining him I found that his spleen was slightly
enlarged, while the lower margin of his liver could just be felt on deep inspira-
tion below the costal margin. On palpating his abdomen he complained or great
pain all over the region of his large intestine. It was noticed that he was a
man of good physique and, although there was evidence of considerable wasting
particularly in his arms and legs, his body was well covered ; his general condi-
tion was favourable, and there was every chance of his recovering from this
acute attack of dysentery. His motions were foul smelling and chiefly consisted
of green sloughs with blood and mucus. His breath had that characteristic
foetid odour, so often noticed at this stage of the disease. On January 27th
another blood film was examined and thirty-one parasites were found. On this
day a large number of the first nymphal instar of Cimex rotundatus, contained
in two tubes, A and B, were fed on the patient's arm. He was not seen again
till the 29th, when a blood film was taken and 100 parasites were found. On
the 30th the two tubes of nymphs, A and B, were again applied and, most of
them fed for the second time ; a fresh batch of nymphs of Cimex lectularius,
tube C, were fed for the first time as well as a large number of nymphs of
Conorrhinus rubrofasciatus (tube D). On the 30th it was noticed that the
patient was decidedly worse, he was much weaker, and was passing his motions
in bed. On the 31st a blood film was taken and 250 parasites were found, and
on this day the bugs in tubes A, B, C and D were again fed, as well as those in
tube E, a fresh batch of Cimex lectularius. Late in the same evening the
patient became rapidly worse and died the next day.
15
It is important to note exactly when the several batches of bugs were fed.
Two tubes A and B, containing nymphs of Cirnex rotundatus, were first fed on
R. on January 27th, again on the 30th and 31st. One tube C, containing
Cimex lectularius, was applied on the 30th, and. again on the 31st. The nymphs
of Conorrhinus rubrofasciatus, contained in tube D, were first fed on the 30th,
and again on the 31st, while the nymphs Cimex lectularius in tube E were fed
only once on the 31st. It should again be noted that all these bugs were bred in
the laboratory, and that they had their first feed on the patient. It will be seen
then that these experiments were carried out with the utmost precision, and
under natural conditions.
In order to explain more clearly the results which have followed these feed-
ing experiments, it is necessary to again refer to my previous work on this sub-
ject. It will be remembered that in 1907 I repeatedly fed bugs on one or more
cases for several days, and then dissected them after varying intervals. In ex-
periment 1, noted on page 13 of my first report, bugs were fed on case 11 on
March 29th, again on the 30th, 31st and April 2nd. On the latter clay one bug
was dissected and its midgut was examined in the, fresh condition, and a flagel-
late was found, but I then noted that its movements were slow and in no way
similar to that of the natural herpetomonads of mosquitoes. I was then not
aware of the significance of these slow movements of the flagellate stage of the
parasite in the bug, as compared with those of the mosquito herpetomonads.
In experiment 1, case 12 noted on page 14 in the same report, twenty-two
bugs were fed on the case on May 16th ; they were again fed on the 17th and
18th, and on the 19th one bug was dissected, and in its miclgut contents, two
parasites were found which had probably been ingested on the 18th. The
remaining bugs were next fed daily from the 19th to the 31st, and on June 1st
one bug was dissected, which had last fed on the 31st, and fifteen parasites were
found in the film ; some of these showed the earliest changes towards develop-
ment. On June 4th another female bug was dissected, and a single parasite was
found which had still further developed. On June 4th the remaining bugs were
again fed on another case of Kala Azar, and on dissecting four bugs on the same
day, no parasites were found in them. On June 5th another bug was dissected
and eleven parasites were found, but unfortunately no note was then made as to
whether this bug had fed on the previous day. The remaining bugs were
dissected on the 5th and 6th with negative results. In the last experiment
recorded in my first report, six bugs were fed three times on a case and then
five times on a monkey ; on dissecting these bugs three unchanged parasites
were found, but no developmental forms. In the experiment recorded in my
second report, twelve adult bugs were fed on the case, and the last bug was
dissected on the fifth day, none of the bugs having been fed again in the interval.
16
At this stage of my experimental work, I was under the impression that the
parasite, after flagellating in its invertebrate host, passed back to man in the
form of the spirilla-like parasites described by Leishman ; I believed. this would
either take place by regurgitation or by the entrance of these forms into the
salivary glands of the bug. Since then I have learnt that this view was entirely
erroneous, and that the spirilla-like forms are not the final stage in the extra-
corporeal life history of the parasite. By studying the life histories of many
insect herpetomonads, I came to the conclusion that, in order for this human
parasite to find its way back to man, it must be injected into his body in a non-
flagellated form, the post-flagellate stage. In my earlier experiments I aimed
at obtaining a massive infection by feeding my bugs repeatedly, so that I could
the more readily find the infective stages either in the midgut of the bug, or in
its salivary glands ; but, as will be seen later, by the very methods employed, I
failed to prove anything more than that the parasite flagellates in the bug.
Later it was thought that one feed was sufficient, and that if the bags were fed
again the infective stage might be injected into the next person the bug fed on.
Here again I failed. to prove anything more, and only found that the develop-
ment in the bug may go on up to the fifth day without spewing the infective
forms to be the predominating ones.
Though convinced that the bug was the true invertebrate host, I was dis-
satisfied with the results obtained, and felt that there was some fact in connec-
tion with the development of the parasite in the bug which was yet to be dis-
covered. On turning my attention to the method of the spread of Kala Azar
in Madras, I could not help thinking there was some natural obstacle which
frequently prevented the parasite from completing its life history in the bug ;.
this was the only satisfactory explanation of why the disease does not tend to
spread outside the endemic area. Precisely the same was noted in the case of
the parasite of Oriental Sore in Cambay, and, as a result of my work on this.
parasite, I came to the conclusion that the natural obstacle was connected with
the digestion of the blood in the bug ; for instance it was soon found out that
temperature had a marked effect on the digestive processes in this insect. It
was then thought that the problem might be solved by studying the effect of
blood on some natural herpetomonas of a blood-sucking insect. Such a para
site was found in Herpetomonas culicis, parasitic in Culex fatigaizs, and it was
noticed that the parasite could be readily found in the male mosquito, but
rarely in the female. On investigating this question further, it was found
that when female mosquitoes were bred out from larvae, the majority of them
were infected, and when they were fed on human blood, the parasites almost
invariably disappeared, in a few however they were recovered. Although a
satisfactory explanation of this disappearance of the flagellates was not at once
17
forthcoming, it appeared as if the blood had some injurious effect on the flagel-
late stage of the parasite. I had just finished these experiments with the mos-
quito herpetomonas, when I heard of the suitable case of Kala Azar in the .
Hospital and bugs were fed as noted above.
On February 1st, 1912, one of the nymphs of Cimex rotundatus, from tube
A, was dissected ; this bug had first fed on H. on January 27th, and for the last
time on the 30th. In the smear, made from its midget contents, the early
stages of the development of the parasite were found consisting chiefly of free
parasites and several shewing the commencing formation of the flagellum. On
the same day a nymph of Cimex lectularius from tube E was also dissected,
this bug had fed only once on R. In the film, much the same parasites were
seen, except that the multiplication appeared to have gone on more rapidly, for
several young flagellates were found. On February 2nd another nymph of
Cimex rotundatus, tube A, was examined, and now all the parasites were well
advanced towards flagellation, and there were many mature flagellates. On
February 3rd I decided to give the bugs in tubes A, B and C, another oppor-
tunity of feeding. A case of Kala Azar was selected, in whose peripheral blood
there were only a few parasites to be found, six were seen in a blood film on
this particular day. About half of the bugs in the several tubes fed well on
this case. On February 4th one of the nymphs from tube B was examined,
it had not fed on. the second case on the 3rd. In the film a number of flagel-
lates as well as younger stages were seen. On February 5th a specimen of
Cimex lectularius from tube C was examined, and a large number of mature
flagellates, as well as many shewing multiple segmentation were found. On
the same day one of the nymphs, C. rotundatus, which had fed on the second
case on the 3rd was examined, and except for two unchanged parasites none of
the early developmental forms or flagellates were seen. A number of other
specimens of both species, which had fed on the second case on the 3rd, were
next examined and it was at once found that all the parasites had disappeared
and only the ghosts of some flagellates could be recognised. Several bugs,
which had not fed on the second case, were then examined and in them all, the
further developmental forms were readily found, and in several of the bugs an
enormous multiplication had taken place, and masses of rosettes were seen. It
was at once recognised that the long suspected natural obstacle was discovered,
and it was now quite clear that the younger developmental forms and the
flagellates, which may be present in a bug, are destroyed when it takes a fresh
feed of blood; in this case it was the blood from a patient suffering from Kala
Azar. Two nymphs of Cimex lectularius, tube B, were next fed on a young
monkey, Macacus Sinicus, and they were examined exactly twelve hours later.
Here again all the developmental forms, which had been unmistakably present
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in these two bugs, were completely destroyed, and many groups of flagellates
could just be recognised as such. There could. now be no possible doubt that
there is some body, present in man's blood, as well as in that of the monkey,
which is inimical to the development of the parasite.
This discovery at once explains some of the most puzzling questions in
connection with the disease and its parasite. Looking back at my previous.
attempts at infecting the bug, it will be seen that, by repeatedly feeding the in-
sects, I was only destroying any developmental forms already present, and that
in this way massive infection of a bug could never be produced ; the failure to
attain this object was at the time most disappointing. Had I only kept the bugs.
utilised. in the experiment recorded in my second report five days longer, it is.
very probable that I would then have discovered the complete development of
the parasite.
This new discovery explains another point in connection with the parasite
in man, which so far as I am aware no one has yet attempted to solve. Why
is it that the parasite of Kala Azar never becomes a flagellate in man's blood,
or his organs ? There must be many occasions when it is free in the blood, and
it should then develop into a flagellate. Further, it is evident that the white
blood cells are incapable of destroying the parasite, this being the case there is
no reason why it should not enlarge and become a flagellate while still in the
cell. This difficult question is I believe at once explained, by the discovery thai
human blood has some body in it which not only prevents the process of flagella-
tion, but also destroys the flagellates. This substance is in all probability the com-
plement, and it is well known that it is itself destroyed in about two days, when
blood is drawn from the human body. This fact further explains why the
parasite only begins its development in the bug on the second day. It may
also explain why Kala Azar of the Mediterranean littoral only attacks young
children and is rarely met with in adults. Lastly this discovery offers, in my
opinion, the only satisfactory explanation of the curious fact that Kala Azar
in Madras hardly, if ever, spreads outside its endemic area. Assuming that Kala
Azar is transmitted from man to man by some blood-sucking insect, why should
the disease cling to one spot in a town and not spread, as do other insect-borne
diseases ? It is only right that an attempt should be made to answer this
question. There must be innumerable opportunities for the true invertebrate
host to become infected in large numbers, and there is no apparent reason why
they should not carry the parasite outside Madras. This puzzling fact in
connection with the disease can now be better explained, when it is known that.
though many bugs may become infected, only those which do not feed again
till the parasite has passed back to its post-flagellate stage are infective ; the
number capable of transmitting the parasite must then necessarily be reduced.
19
Exactly the same will in all probability hold good for the parasite of Oriental
Sore, for as I observed the disease in Cambay, I was struck by the fact that
it has never spread outside the town itself.
I will now describe the life history of the parasite as I have studied it in
Cimex rotundatus and Cimex lectularius fed only once on the patient.
As already stated, the bugs were kept in glass tubes, and throughout the
experiment were placed in a cold incubator at 22° to 24°C. They were dissected
in the way I have already described, and the films when smeared out, were fixed
with absolute alcohol and stained with Romanowsky's stain.
The Pre-flagellate Stage in the Bug.
The parasite is ingested by the bug, either in a white blood cell, or in a
macrophage, and it remains in an unchanged condition from about 36 to 48
hours. Figures 1, 2, 3, and 4, on the plate, depict several parasites in white
blood cells from the bug ; the leucocytes in each case shew commencing dis-
integration as evidenced by the vacuoles seen in their protoplasm. While still
lying in a leucocyte, the parasite may shew the earliest developmental changes,
it enlarges, its protoplasm becomes vacuolated, granular and more voluminous
and now takes on a marked blue colour when stained with Romanowsky's stain.
At the same time the nucleus enlarges and stains a lighter pink, and may con-
tain a single central chromatic granule, or a number scattered about. The
nucleus at this stage may lie at one end of the parasite, or about its centre. The
blepharoplast is now distinctly enlarged, and may lie close up to the nucleus or
more towards the periphery of the cell. The parasite at this stage usually retains
its original shape, and measures from 4 to 6 or 7 µ in length, and from 3 to
4 or 5 µ in breadth. Figure 5 is a young growing parasite still lying in a
leucocyte, while figure 6 represents a free form and figure 7 another large one
lying at the margin of a disintegrating white cell. When looking over a film
made from the midget contents of a bug, these early developing forms can at
once be recognised, they are extremely characteristic, their size, together with
their granular blue staining protoplasm, render them very striking objects, and
they cannot possibly be mistaken for anything else. Before enlarging further,
many of these young forms exhibit the earliest signs of binary fission, their
nuclei become kidney shaped and the chromatic granules may be seen grouped
together in two halves. Figures 8, 9 and 10 shew this appearance, while in
Figure 11 although the nucleus is still more or less round, the blepharoplast is
elongated and thickened. As further growth takes place, the nucleus may
divide, as seen in Figure 12, or the blepharoplast may first enlarge and then
divide, Figure 13. At this stage the parasite itself may exhibit the early
20
appearances preceding simple binary fission ; its body becomes indented, usually
at the end furthest away from the nucleus, and. it later splits into two.
The Flagellate Stage.
It will be remembered that, in my second report, I pointed out that at this
stage of the life history of the parasite, one or other of two changes may take
place ; (1) the parasites may rapidly pass on to flagellation ; or (2) what is
more important, they further enlarge and by consecutive division of their nuclei
and blepharoplasts produce a rosette of from four to eight parasites. I will
first follow the changes undergone by the single forms as they approach flagel-
lation. About the time the nuclei are going to divide, there appears a small
pink staining area between the blepharoplast and the margin of the cell ; it is
from this structure that the flagellum is developed. In my second report, I
considered it was of the nature of a vacuole, and that the flagellum grows out of
it as evidenced by the pink staining strands seen lying across it. Since then
I have made a special study of the formation of the flagellum, not only in this
parasite, but in the allied parasite of Oriental Sore and in many natural herpe-
tomonads, and am able to give a more satisfactory explanation of its nature.
From a careful study of the formation of the flagellum at its earliest stage, it
was seen to grow out from an already existing pink staining filament. Any
one who has studied deeply stained specimens of the parasite of Kala Azar will
have noticed that many of them have a pink filament close to, but not touch-
ing the blepharoplast, and which stretches towards the periphery of the cell.
This pink filament may be straight or slightly bent, and usually lies perpendi-
cular to the blepharoplast. Christophers was the first observer to draw atten-
tion to this filament and he has clearly depicted it. Since then several
observers have described it from the parasite of infantile Kala Azar and that of
Oriental Sore. Novy speaks of it as the rhizoplast.
In many of the bugs, both Cimex rotundatus and Cimex lectularius, I
have had the opportunity of examining a large number of parasites exhibiting
the earliest growth of the flagellum. Figure 1S depicts a parasite in which the
pink filament is merely thickened, while figures 12, 13, 14, 15 and 19 shew the
various appearances just prior to the extrusion of the flagellum. As the pink
band. thickens more and more, it appears as a round or oval area, and extending
across it there may be one or more thin filaments. This vacuole-like appear-
ance is I believe nothing more than the enlarged filament stretched out as the
parasite adheres to the slide. Later, the flagellum, which appears to consist
of several delicate filaments, protrudes from the body of the parasite and then
becomes free. The question now arises, what is the nature of the pink staining