NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
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they found 6.2 per cent, of the mosquitoes to be infected with Crithidia alone,
and. 6 per cent. with Herpetomonas, while 2.1 per cent. contained both Crithidia
and Herpetomonas. It is most important to note that mosquitoes may be
infected with Herpetomonas as well as with Crithidia. The writer has record.-
ed the presence of these flagellates in Culex fatigans, Cuiex sp. and Neocellia
stephensi, in Madras ; Adie has found them in several species of anophele s
mosquitoes in North India. Lastly Wenyon describes a herpetomonas from the
malpighian tubes of the larvæ of Stegomyia fasciata in Bagdad.
It is unnecessary to make any further reference to Schaudinn's observations
on the development of the blood. parasites of the little owl in Culex pipiens,
they have been fully discussed by Novy as well as by the writer.
Now let us suppose that an investigator in Madras, suspecting that
the parasite of Kala Azar is transmitted by some species of mosquito,
collects a large number of the common forms, either by catching the adult
insects, or by breeding them out from the larvæ. Let us suppose, for instance,
he selects Culex fatigans, Neocellia stephensi, and Myzomyia rossi. After
feeding these mosquitoes on Kala Azar patients he would most certainly
find flagellates in a varying percentage of those he examined ; and. would
naturally conclude that they represented the developmental forms of the human
parasite. This is exactly the conclusion the writer came to in 1905. Precisely
the same results would be obtained if the observer fed the mosquitoes on cul-
tures of the human parasite ; here however the mosquito may at the same time
contain three different species of flagellates in its stomach, a possible double
infection of herpetomonas and. crithidia, as well as the flagellate of the parasite
of Kala Azar. When the mosquitoes were only fed on the peripheral blood
there would be less chance of their containing the flagellates of the human
parasite.
The observer who only has a slight acquaintance with the flagellates of
mosquitoes would. be further led. astray on finding that there was a great varia-
tion in the number of infected mosquitoes (females). He would, for instance, find
that in a batch of mosquitoes caught, say in a particular well or house, or bred.
out from larvæ collected. from a certain tank, pond. or well, that not a single mos-
quito after being fed. on the Kala Azar patient contained flagellates ; and might
conclude that this species was not the suitable host of the human parasite, or
that none of the female mosquitoes sucked up parasites. On the other hand. he
may find almost every female mosquito infected, and if it was of a different
species, he may conclude that he had at last found the true invertebrate host of
the parasite ; the explanation would. most probably be, that this particular
mosquito was taken from a well where it readily becomes infected with its
herpetomonas or crithidia. The fact that a particular mosquito, when collected
5
from a well, is nearly always infected with flagellates, and that the same
mosquito bred out from larvæ from a tank is never infected is of the first
importance in connection with such feeding experiments.
If the observer carried his observations further he would. find. that an infect-
ed. mosquito often passes out the flagellates in its excreta, and that in addition
to the long mature forms he would find. many short and. round forms, closely
simulating similar stages of the parasite of Kala Azar. In the mid-guts of the
mosquitoes he would. find. flagellates, as well as non-flagellated forms, and all the
intermediate stages between the two. In some of the mosquitoes he would. find
only a few parasites, while. in others he would find vast numbers. All these
observations would support his view that the parasites were none other than
developing parasites of Kala Azar.
In a recent paper Franchini claims to have observed the multiplication
of the parasite of Italian Kala Azar in the alimentary tracts of Anopheles
maculipennis and. Stegomyia fasciata. In this case the mosquitoes were fed.
on cultures of the human parasite, and were examined at varying intervals.
From half to two hours after the mosquitoes fed, flagellates were abundant, but
after eight hours they became rare, and then appeared more globular. Twelve
hours after the insects fed, fewer parasites were found, and they were now
without flagella. After an interval of from twenty-four to forty-eight hours, the-
parasites had all rounded up and were exactly similar to the characteristic intra-
cellular stage of the parasite of Kala Azar. Franchini concludes that the
flagellated stage of the human parasite can live, and develop, in these mos-
quitoes, and that it passes on to its round or oval form, which is then ready to
be transmitted to man. It is not quite clear where these various stages were
found, but presumably the rounding up forms were most abundant in the hind
guts of the insects. Franchini makes the extraordinary statement that, in spite
of the presence of bacteria in the mosquitoes, the flagellates persisted and con-
tinued to develop. It is well known that the parasite of Indian Kala Azar and
that of Italian Kala Azar will never flagellate in any medium infected with
bacteria, it is therefore difficult to understand how they were able to maintain
themselves in these mosquitoes.
In a still more recent paper Franchini again draws attention to his earlier.
observations, this time depicting the various stages of the parasites. In view
of my failures to infect mosquitoes with the parasite of Indian Kala Azar, and
knowing that many species are infected with very similar flagellates I find it
necessary to carefully analyse Franchini's observations. At the outset he
states he has been unable to recover any stages of the parasite of Italian Kala
Azar in the alimentary tracts of Cimex lectularius, Ctenocephalus canis (P.
serraticeps) and Pulex irritans though they had fed on cultures containing
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numerous parasites, similar results were obtained in the case of P e d i c u l u s
capitis. Several hundred experiments were carried out with Anopheles
maculipennis fed on cultures, and numerous controls were examined. He states that
the parasites found. in the mosquitoes fed on cultures, could not be mistaken for
any natural flagellates they may contain, for the latter can be distinguished by
their nuclei, different staining and much larger size. After years of study of
the flagellates of mosquitoes, especially Herpetomonas culicis, I -would feel ex-
tremely diffident in expressing an opinion as to the true nature of a herpe-
tomonad occurring in a stained smear of the midgut of any mosquito which
had fed on cultures of the parasite of Kala Azar. I must confess I know of no
differences in the structure of their nuclei or of their reactions to stains, espe-
cially to Romanowsky's or Giemsa's stain. It is true that, in the majority of
instances, the flagellates of mosquitoes are larger than the corresponding stage
of the human parasite. Franchini's diagrams however do not in my opinion
bear out even this point. In his plate 2, the figures are said to be drawn with
a Zeiss objective 6, and compensating ocular 8, presumably with a camera
lucida, though this is not stated ; this would represent a magnification of about
500 diameters. The longest mature flagellate depicted in Figure I may
well be compared with figure 15 in the plate accompanying this paper.
My figures of the mature flagellate stage of Herpetomonas culicis were drawn
by the aid. of a camera lucida with a Leitz 1/16 oil immersion and compensat-
ing ocular 8 and are therefore magnified about 1260 diameters. The flagellate
noted in Franchini's plate 2, proves then, on comparison, to be the same size
as that depicted in figure 15, and only slightly shorter than the very long forms,
figures 30 to 37. Precisely the same will be noted on comparing the figures of
the round forms of the parasite from A. maculipennis with the round forms
depicted in my plate. This would be exactly in accordance with my observa-
tions, for as stated above though most of the flagellated forms of H culicis are
larger than the corresponding stage of the parasite of Indian Kala Azar, many
are actually smaller.
It is evident that Franchini has encountered the natural flagellates of
maculipennis, for he states that they are rather scarce; he does not however
mention whether his mosquitoes were bred from larvæ, or caught as mature
insects. Nor does he tell us whether these natural flagellates of A . maculi-
pennis were commoner in certain batches of these insects, and rare in
others ; points which I have laid stress on above. There can be very little
doubt that Franchini has actually seen the flagellated forms of the parasite of
kala Azar in the mosquitoes, but the proof that all the forms he depicts, are
those of the human parasite, are in my opinion based on very slender evidence.
It is unfortunate that no precise statements are made as to the exact part of
7
the alimentary, tract in which they were found. ,The nature of a herpetomonad,
based. on staining reactions, will not hold. in the case of these parasites, neither
are structural details of much value. A parasite from the alimentary tract of a
blood sucking insect, be it exactly like the parasite of Kala Azar as it occurs in
man, is no proof as to its true nature, yet I find Franchini relies to a great
extent on this point. The granular structure as depicted by Franchini in the
parasites of A. maculipennis are extremely well seen in H. culicis as will be
seen on referring to my figures of this flagellate. Karyokinetic figures in the
nuclei of the parasites of Kala Azar as seen by Franchini, are just as well seen
in the nuclei of Herpetomonas culicis, so that these appearances cannot be used.
in distinguishing the two parasites. It is definitely known that both Anopheles
maculipennis and Stegomyia are naturally infected with flagellates, and I can
find no proof in Franchini's paper that he has conclusively excluded the
possible presence of these parasites in his mosquitoes. I may, also point out
that control experiments are really of very little value, because the number of
infected mosquitoes varies according to the opportunities their larvae have of
becoming infected. I have repeatedly proved this in the case of H e r p e t o m o n a s
culicis in Culex fatigans.I n s p e c i m e n s o f t h i s m o s q u i t o , b r e d o u t f r o m l a r v æ
collected from tanks, I have never found this flagellate, whereas out of those
hatching from larvæ breeding in wells, from 50 to 90 per cent., are usually
infected.
I am therefore of opinion that though Franchini undoubtedly has seen the
parasites of Italian Kala Azar in his mosquitoes, he has omitted to rigidly
exclude other flagellates, and that these forms were present in the hind guts and
fæces of his insects in their post-flagellate stages this stage is extremely like
the human parasite in every way. Whether the parasite of Italian Kala
Azar will actually pass from its pre-flagellate to its flagellate stage, after
being ingested by the mosquito in a leucocyte from the peripheral blood of a
patient, has yet to be demonstrated ; herein will lie the proof of the mosquito,
.transmission hypothesis.
Material and. Methods.
The Herpetomonas which forms the subject of this paper is parasitic in the
.alimentary tract of the larva, nymph, and imago of Culex fatigans, and it is
in all probability identical with a similar flagellate, described from America by
Novy and his Collaborators. I therefore propose adopting the specific name
culicis. There can be little doubt that this herpetomonas was encountered by
Ross when conducting his classical researches into malaria, and he has already
described some of its stages. Culex fatigans is abundant in Madras at all times
8
of the year, and breeds in wells and tanks ; it is particularly fond. of breeding
in the septic tank of this Institute, and over 95 per cent. of the larvæ taken
from this situation were found to be infected. With this rich material close at
hand, there was no difficulty in working out its complete life history.
A large number of larvæ, of all ages, were collected, and placed in large
stoneware trays, and on selecting a particular specimen, its alimentary tract
was dissected out in .6 per cent. saline solution on a glass slide, and then
examined with a No. 3 objective. After a time it became possible to detect in-
fected larvæ by examining their alimentary tracts with a low objective, for the
parasites are only found at the junction of the malpighian tubes with the
mid-gut, and if infected, this portion has a characteristic glistening white appear-
ance. It was also found that the malpighian tubes of an infected larva were
never swollen, and packed with granules, but were shrunken and contained
fewer granules. On dissecting out the alimentary tract, that portion of it con-
taining the parasites was transferred to a clean slide in a drop of saline
solution, and before placing a cover slip over it, a hair was fixed at the side and
the cover slip edged with vaseline ; the parasites could then be examined in the
fresh condition, and as the hair prevented. undue pressure they could be studied
for a considerable time without the gut rupturing. If it was desired to stain
them, the part containing the parasites was still further isolated, the malpighian
tubes being carefully removed, the alimentary tract was then teased up with fine
needles by the aid of a dissecting microscope. The emulsion containing the
parasites was next exposed to the vapour of osmic acid, and with a small piece
of square cover glass it was lightly smeared out on a clean slide, immediately
fixed with absolute alcohol, and later stained with Romanowsky's stain ; the
same technique was employed in the case of the parasites in the nymph and
imago.
The adult mosquitoes were also bred out and were fed on bananas and
human blood, and the behaviour of the parasites under these conditions was.
studied. There can be no question as to the origin of this herpetomonas, for the
larvæ could obtain the parasites from no other source than the water in which
they were living. In the tank mentioned above, many thousands of mosquitoes.
were resting just above the surface of the water, and would naturally pass out
their excreta on to it, and the larvæ when feeding could not do otherwise than
ingest the postflagellate stages. It is interesting to note that the mature flagel-
lated. form of the parasite was never seen in the larvæ, yet the mosquitoes fre-
quently pass out this stage in their excreta ; the flagellates do not appear to be
able to live in the water or in the alimentary tracts of the larvæ. The intes-
tines of the larvæ frequently swarm with bacteria, and. this would probably
explain the absence of the flagellated forms in them. It is true that the pre-
9
flagellate stage may flagellate in the larval gut, but this only occurs in the
mature larvæ, which have by this time cleared themselves of most of the
bacteria.
Two other mosquitoes, Culex concolor, and a species of Joblotia, which
were breeding in the same tank, were never infected with Herpetomonas
culicis ; the larvæ of the former were feeding on the larvæ of Culex
fatigans, and they must have ingested the pre-flagellate stage, yet the parasite
was never found in them. This fact points to Culex fatigans being the
specific host of this parasite in Madras.
There is a small fly, Ephrydid Sp., which is commonly found on the surface
of stagnant water in Madras, and it was abundant in the septic tank ; I have
long known that it is infected with a species of herpetomonas, but I am
unable at present to say whether it is identical with Herpetomonas culicis.
A large number of experiments would have to be carried out before this ques-
tion could be decided ; owing to the great pleomorphism exhibited by these
insect flagellates, and also to the fact that many of them are indistinguishable,
it is futile to attempt to make any definite statement on this point. I would
however like to point out that accurate experiments in this direction would
undoubtedly help towards settling the question, as to whether these flagellates
occurring in different insects belong to one and the same species. I have
already carried out some experiments to try and see whether Herpetomonas
muscæ domesticæ, which is parasitic in Musca nebulo, Will live and develop
equally well in Lucilia serenissima, but have found that the pre-flagellates and
flagellates of this herpetomonas are unable to live in the alimentary tract
of this fly ; both Musca nebulo and Lucilia serenissima feed side by side on the
meat in the bazaar shops. In the same way the herpetomonas of Lucilia
serenissima will not live in the alimentary tract of Musca nebulo ; all these
experiments were carried out with bred flies. These observations will be
referred to in detail in the papers dealing with these parasites.
THE STRUCTURE AND LIFE HISTORY OF THE PARASITE.
The Pre-flagellate Stage.
The pre-flagellate stage of Herpetomonas culicis is almost entirely con
f i n e d t o t h e l a r v a l g u t , b u t m a y a l s o b e f o u n d i n t h e n y m p h , a n d e v e n i n t h e
imago ; it is in this latter situation that this stage may lead the observer astray.
It was pointed out that on dissecting out the mid and hind gut, the parasites
will be seen massed together, and under a dissecting lens they appear as a
10
glistening patch, just posterior to the junction of the malpighian tubes
with the mid-gut ; they were never seen in the urinary tubules,
On examining the mass of parasites under a 1-12 objective in the fresh
condition the individual parasites can be readily studied ; they appear as
round, oval, or elongated bodies, depending upon their degree of development,
the elongated forms represent the gregarine or resting stage of earlier authors.
Their protoplasm may contain many granules grouped together at one or other
end ; their nuclei appear round, and frequently the small rod-shaped blepharo-
plasts may be detected. If such a group of pre-flagellates is watched for
some time, the individual parasites will be seen dividing, a process Which may
occupy an hour or more. There can be little doubt that these young forms
adhere together by some sticky substance, for considerable pressure is required
to dislodge them from the intestinal wall, and when this is accomplished, two,
four, or more, may be seen adhering together, and the coverslip must be
further compressed before the individual parasites can be separated. It is
very probable that this secretion is necessary in order that the parasites may
remain attached to the intestinal wall, otherwise they might be expelled by
the powerful peristaltic waves which pass along the mid and hind gut.
Text Fig. I.
In the medium sized. and mature larvæ, the parasites may be seen in
large numbers packed together in many layers, so that they extend into the
upper part of the hind gut. Text Fig. I. is a diagramatic illustration of
an infected larval gut as seen under a low power. On smearing out the
11
part of the gut containing the parasites, and staining it with. Romanowsky's
stain, all the changes, undergone by the parasites during the pre-flagellate stage,
can be well studied ; I will now describe the minute structure of these forms.
Figure I. on the plate represents the early pre-flagellate stage, which
is found in the small larvæ. These forms measure about 4 α in breadth and
are almost globular in shape ; their protoplasm stains blue, is vacuolated,
and may contain a number of large, dark pink staining granules, either scattered,
or close together. The outer layer of the protoplasm, or ectoplasm is of the
nature of a closely fitting sheath. The nuclei of these forms are as a rule
situated centrally, but may lie to one or other side ; they stain a light pink,
and may contain a dark central granule or a number grouped together. The
nuclei frequently shew a well marked nuclear membrane. Lying at some
little distance from the nucleus will be seen the rod-shaped blepharoplast.
In deeply stained specimens, and more particularly in those which have
ruptured, a delicate pink filament may be seen arising close to the blepharo-
plast and stretching across the cell. This is the flagellar myoneme, and it
is from this structure that the flagellum arises later on ; it has been noted
in my paper on Herpetomonas donovani.
The pink staining granules noted above are clearly connected with the
growth of the parasite, for if infected larvæ are placed in clean water for
several days, and the parasites they contain are then examined, the granules
will be found to be fewer in number, and may even disappear altogether.
They are therefore of the nature of food granules, but as there is no satisfac-
tory means of analysing them chemically I am unable to express a definite
opinion regarding their true nature.
In the fresh condition, these young parasites do not shew any movement,
their shape remains the same, although as growth proceeds, they slowly enlarge,
their protoplasm becoming more voluminous and they now exhibit the
changes preceding simple biniary fission, figures 2 and 3. Their nuclei at
this time increase in size, and appear more granular, they then become dum-
bell shaped, figure 4. At the same time their blepharoplasts elongate, pre-
paratory to division. Later the nuclei divide and the two halves separate,
and this is followed by the division of the blepharoplast, figures 5, 6 and 7.
The parasites now become constricted at one end, and later spilt into two,
and many of them may be seen still attached at one or other pole. This
process of division appears to be the common one, and as it is repeated several
times, the parasites become massed together in layers. They adhere to each
other, as well as to the intestinal wall. A careful search was made as to
whether the parasites ever became intracellular, but they were never seen
inside the intestinal cells.
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