NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
5
STEGOMYIA SUGENS.
Thirty-one female mosquitoes, fed several times on a suitable case, were
dissected at regular intervals but with negative results ; this mosquito does not
as far as I know contain a natural flagellate.
EXPERIMENTS WITH TICKS. ORNITHODORUS SAVIGNYI.
Several adults and nymphs were repeatedly fed on a case of Kala Azar
shewing parasites in the peripheral blood, and were dissected with negative
results.
EXPERIMENTS WITH CIMEX ROTUNDATUS.
Before describing these experiments, it is again necessary to state that this
bug never contains, as far as I know, a natural flagellate other than that which
develops from the parasites of Kala Azar or Oriental Sore. Bugs, both
nymphs and adults, were repeatedly fed on cases of Kala Azar in whose peri-
pheral blood parasites were either numerous or scanty. They were dissected at
varying intervals after their last feed, and in a few of these bugs the parasite
was recovered in an unchanged condition in a leucocyte, as well as free forms
shewing some changes towards flagellation. These experiments were carried
out at a temperature of from 80° to 82°F. In my second report I described
some further experiments with Cimex rotundatus . Twelve adult bugs were fed
on a case in whose peripheral blood 359 parasites were found in one film, and
on dissecting them at varying intervals up to the firth day, all the forms, from
the unchanged parasite up to the fully developed flagellates, were recovered from
the midguts of the bugs. The following is a summary 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 macro-
nuclei enlarge and shew 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 micro-nuclei, may result in the formation of rosettes.
The single flagellates enlarge and begin to divide longitudinally to result in oval
or spindle-shaped cells ; while the rosettes after flagellation begin to divide up
into separate elongated flagellates. The oval or spindle shaped parasites divide
repeatedly either by equal or unequal longitudinal 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, longitudinal division of the oval and irregular
flagellates progresses rapidly, so that by the fifth day the majority have become
small or spirilla-like flagellates." In connection with these experiments it is
necessary to draw attention to the fact that in the first series, the bugs were
repeatedly fed, whereas in the last they were only fed once, and the last bug was
B 2
6
dissected on the fifth day. My reason for drawing attention to the number of
times the bugs were fed will be seen later.
Donovan has attempted to confirm my observations on the development of
the parasite in Cimex rotundatus. On three occasions he fed bugs on suitable
cases, but states that he found no developmental changes in the parasites
ingested, even after a week or ten days. He gives no precise details of these
experiments. Being dissatisfied with his attempts to infect Cimex rotundatus
he has searched for some other blood-sucking insect of local distribution, and
one which is not so ubiquitous as the bed bug. He has succeeded in finding
Conorrhinus rubrofasciatus de Geer, which he considers to be the most likely
transmitter of the disease. He notes that both male and female conorrhini can
be found which contain either digested or fresh mammalian blood in their
midguts, and that both the adults and nymphs will readily suck up human
blood. He records a flagellate of the genus Crithidia (C. conorrhini, Donovan)
from the alimentary tract of the adult insect. He has fed this insect on suit-
able cases of Kala Azar, but has been unable to find any of the developmental
forms of the parasite, and is now inclined to abandon the idea that it is the
natural carrier. In connection with this insect I may point out that I have
fed on myself the adult insect and several of the nymphal instars, and have
found that the bite is most irritating. The actual puncture made by its
proboscis is felt as a sharp sting, and about 12 hours later a large erythematous
patch about the size of a shilling appears at the site of the bite ; it remains in
an inflamed and irritable condition for about a fortnight and then fades away,
and a small nodule the size of a No. 6 shot persists for about one month. I
therefore feel certain that were this bug a common blood-sucker in the Indian
houses in George Town, it could hardly escape detection ; further, the marked
symptoms resulting from its bite could not possibly escape observation. In 1906
when I visited the houses of people suffering from Kala Azar, on enquiring
what insects were in the habit of biting them, I never even heard of this
Conorrhinus ; it is a large coloured insect, and if it were a regular blood-sucker
it would surely be well known. Recently I have endeavoured through several
medical friends to obtain some information regarding its distribution in George
Town, and as a result I have obtained a single specimen. On further enquiry I
found that it never bites people, but is attracted by a light and will thus enter a
house. I have however now had the opportunity of carrying out some feeding
experiments with this bug, and these observations are described further on.
This concludes the summary of all the experimental work which has been
carried out in India with blood-sucking insects, and I will now give a critical
review of the recent work on the closely allied form of Kala Azar of the Mediter-
ranean littoral.
7
Feeding Experiments with the Parasite of Infantile Kala Azar.
In 1908 Nicolle and Comte made the important discovery that dogs in
Tunis are naturally infected with a parasite indistinguishable from that of
human Kala Azar. As the result of this discovery, Nicolle has advanced the
hypothesis, that the Kala Azar occurring along the Mediterranean littoral is of
canine origin, and that some ectoparasite of the dog is the invertebrate host.
This hypothesis has been further elaborated by Basile who believes that the dog
flea Ctenocephalus canis and probably Pulex irritans transmit the parasite from
dog to dog, from man to man, from dog to man and from man to dog. Basile
supports his statements by the following series of experiments :—
A number of dogs which were born in Rome, and whose bone marrow was
examined for parasites, were taken to Bordonaro, and kept in a house in which
cases of Kala Azar had occurred. Several months later the dogs living
in Bordonaro developed Kala Azar, while the control animals in Rome
remained healthy. Fleas (C. canis) taken from the infected dogs shewed
flagellates in their intestinal tracts. Basile concludes that the dog flea is
the invertebrate host of the parasite of canine Kala Azar. He next
carried out a series of experiments to prove that this flea is the actual
transmitter of the disease. Several fleas were taken from a clean dog, and
placed in glass vessels, and kept at a temperature of 22°C. Later, a few of
these fleas were placed in a vessel containing splenic juice from an infected
dog, while the remaining fleas were kept as controls. The fleas which had been
in contact with the infected material were later dissected, and. an emulsion was
made of the midgut contents of some of the fleas, and it was then injected sub-
cutaneously into a young dog, which Basile states was clean. Smears were
made from the gut contents of the remaining fleas which had fed on the spleen
juice, and on being stained. they were found to contain numerous developmental
forms of the parasite ; the control fleas were all negative. Fifteen days later
the young dog which had been inoculated with the emulsion from the guts of
the fleas developed signs of infection. On the 29th day the dog died, and it was
found to be heavily infected ; Basile concludes that the dog flea, C. canis, is the
invertebrate host of the parasite of canine Kala Azar, and that in the same way
P. irritans will probably be found to be the transmitter of Infantile Kala Azar.
In 1911, continuing his experiments, Basile believes he has succeeded in infect-
ing two young dogs, and an older one, by keeping them in a kennel with a dog
suffering from Kala Azar ; fleas which were abundant on the infected dog
passed over to the experimental ones. Thirty days after the experiment was
begun, hepatic puncture was performed on the three dogs, and they were found
to be infected ; two dogs kept as controls remained healthy. Basile concludes
8
that this experiment strongly suggests that the fleas from the infected dog
carried the infection to the three, presumably, clean ones. In his next experi-
ment Basile records having infected four dogs in Rome, by placing on them
fleas, C. canis, and P. irritans, obtained from Bordonaro. He claims that the
dogs used in this experiment were clean, and that there was no opportunity of
their becoming infected before the experiment was begun. The fleas were
placed on the dogs on December 1st, and early in March they all died of Kala
Azar ; two control dogs remained healthy. He concludes that this rigorous.
experiment proves that canine Kala Azar is transmitted by C. canis and probab
-ly by P. irritans. As the result of the examination of one thousand fleas taken
off dogs, and from the beds of people in Bordonaro, he found four fleas to be
infected with the parasite, and he believes it passes through its extra-corporeal
life history in C. canis. The stained preparations of the intestines of these
fleas shewed the pre-flagellate and post-flagellate stages and that these were
identical with the figures given by Rogers of the cultural forms of the parasite
of Indian Kala Azar. It should here be noted that Basile does not state
whether he found the unchanged parasite in a leucocyte, the degeneration of
the leucocyte resulting in the liberation of the parasite, and the early develop-
ment of the flagellum ; nor does Basile make any precise statements regarding
the part of the intestine of the flea in which the several stages of the parasite
were found. It is important to know for instance where the post-flagellate
stages were encountered, for their exact position in the alimentary tract of the
flea would enable one, who has studied the natural herpetomonads of fleas, to
distinguish them from the pathogenic forms which the flea may ingest.
Another transmission experiment is recorded by the same observer in
collaboration with Visentini. A young dog born in the laboratory was isolated
for about six months, when fleas C. canis and P. irritans from Bordonaro were
placed in large numbers on it. About a month later liver puncture revealed
parasites in small numbers. In still another experiment, Basile records the
examination of fleas P. irritans taken from the bed of a child suffering from
Kala Azar, and in the intestine of one, parasites identical with those previously
seen in C. canis were found ; here again no mention is made of the unchanged.
parasite in a leucocyte. Basile, La Cava, and Visentini, examined fleas taken
from an infected dog living in a house in which there was a case of human Kala
Azar, and found in one of them numerous parasites. Later, fleas were collect-
ed from both dogs and human beings, and were placed on two dogs which were
born in the laboratory, another dog of the same litter being kept as a control.
About three months later the dogs began to shew signs of having contracted the
disease, later still both died and at the autopsies varying numbers of parasites
were found in their organs ; the control dog was again found to be healthy.
9
Basile next proceeded to study the life history of the parasite in C. canis and
P. irritans. He endeavoured to infect them by placing them in contact with
cultures, of both the canine and human parasites, but the only result was the
finding of degenerating flagellates in their midguts. Several other experiments
were carried out by placing the cultural forms on various media, but the results
were totally negative ; that is to say, that though the fleas had, presumably,
ingested the flagellates, these were unable to maintain themselves in the fleas
and soon died. Basile states that this method of infecting fleas is unsatisfac-
tory. He further maintains, that these negative results, as well as those of
Franchini, are not conclusive, but that he has no doubt that the dog flea
C. canis is capable of transmitting the parasite. It is difficult to understand
why, if Basile is sure that the dog flea is the invertebrate host, fleas were not fed
in the natural way on heavily infected dogs, and then dissected, and a search
made for the unchanged parasite, and its development up to the flagellate stage.
Sangiorgi is the next observer who has examined blood-sucking insects for
the developmental forms of the parasite. A large number of bugs, presumably
Cimex lectularius, taken from the houses of people suffering from Kala Azar,
gave negative results. In fleas, C. canis, taken from dogs caught in the town
of Catania, he was able to recover in a single flea a large number of flagellates
resembling the cultural forms of the parasite of canine Kala Azar. He con-
cludes that these are the developmental forms of the canine parasite. Later,
out of 378 fleas collected from dogs, fourteen were found to be infected with
flagellates. In another paper Sangiorgi records the infection of a dog in Turin,
which was contracted by coming in contact with an infected. dog from Tunis,
and in two out of thirty-nine fleas (C. c anis), taken from this dog shortly before
its death, flagellates resembling those previously described by this author, were
found. Sangiorgi considers this transmission experiment to be conclusive.
Basile in his most recent note states that he has found in P. irritans in
Catania, a flagellate identical to that described by Sangiorgi from C. c anis.
Franchini has carried out some experiments with Cimex lectularius, Pulex
irritans, Ctenocephalus canis, and Pediculus capitis. All these insects were
made to feed on cultures of the parasite (presumably of human Kala
Azar), but he states he has failed to find any of the parasites or
their developmental forms in their midguts. He next turned his attention
to mosquitoes, and utilised a species of Anopheles of the claviger variety.
These insects were placed in glass jars, at the bottom of which there was a ten
to fifteen day old culture, which was covered with thick gauze. On dissecting
these mosquitoes, numerous flagellates were found after an interval of
thalf to two hours, and from five to eight hours they were rare. After ten to
welve hours they were seen to have lost their flagella, and alter twenty-four,
10
thirty-six, and forty hours, small round and oval forms were seen, which
Franchini states are identical with the parasite as it occurs in man. These ex-
periments were conducted at a temperature of from 28° to 30°C. Franchini
concludes that the parasite of Kala Azar is able to live and multiply in the
alimentary tract of this species of anopheles, and that the living flagellates may
be passed out in its excreta. He also states that the parasite persisted in the
mosquitoes although they were heavily infected with bacteria, whereas in vitro
these organisms are fatal to its development.
The last observations it is necessary to quote are those of Gabbi, Franchini
and Scordo. In some recent experiments, they state they have failed to infect
both dog and human fleas with the parasite of human Kala Azar, and that the
latter degenerate when placed in culture with the gut contents of these fleas ;
the bacteria, which may be present in the intestinal tracts of fleas, invariably
destroy the parasites. These experiments at once recall those of Basile but the
results are totally divergent. Gabbi has also carried out some further experi-
ments by placing starved fleas in contact with spleen juice from a human case
of Kala Azar, but he has not been able to observe any developmental changes.
Again the examination of fleas taken from the beds of children suffering from
Kala Azar, have failed to shew any flagellates. Gabbi, as a result of these
experiments, doubts whether infantile Kala Azar is of canine origin, and con-
cludes that neither the dog flea or the human flea, play any part in the trans-
mission of canine and infantile Kala Azar.
I have followed these experiments with the greatest interest, and as I have
for some time studied the natural flagellates of fleas, I am in a position to make
some critical remarks regarding the experiments noted above. To any one who
is not intimately acquainted with the many pitfalls which exist in connection
with this work, the transmission experiments which Basile records would lead
them to conclude, that canine Kala Azar and the infantile form are one and
the same disease, and that the dog flea, C. canis, and possibly the human flea,
P. irritans, are the true invertebrate hosts of the parasite.
I will now endeavour to show, that in spite of Basile's apparently conclu-
sive transmission experiments with fleas, there is yet very little convincing
proof that infantile Kala Azar is of canine origin, and that the dog flea is the
true invertebrate host ; in short, the problem of the etiology of both canine and
infantile Kala Azar has yet to be solved. I will first deal with the flagellates
which have been found in the dog and human flea in Italy. It is now univer-
sally recognised that fleas in many parts of the world are infected with natural
flagellates, which have no connection with any blood parasite. A better ex-
ample could not possibly be given than Crithidia pulicis, Porter, whose life
history has been accurately described by Miss Porter ; her experiments were
11
carried out with such precision that there can be no possible doubt regarding
the nature of this flagellate. Unfortunately the complete life history of a flea
herpetomonas is not yet known, though Swingle has described much of the life
history of Herpetomonas pattoni, Swingle. I have had the opportunity of
studying the flagellates of two species of fleas, namely, in Ctenocephalus felis,
the dog, and the cat flea of Madras, and in Ceratophyllus alladinis* the flea
found on the squirrel, Funambulu palmarum. In Ctenocephalus felis, I have
found that there are two distinct types of herpetomonads, one which is exclu-
sively found in the malpighian tubes of the larva and adult, insect, and the
other in the midgut of the larva and mature flea. The species which occurs in
the malpighian tubes of the larva, though it possesses a flagellum is only slightly
motile, while the form which is found in the midgut is highly motile, and is al-
most indistinguishable from the flagellated forms of the parasites of Kala Azar.
In Ceratophyllus alladinis, on the other hand, I have only found the non-motile
form which occurs in the malpighian tubes of the larva and adult insect. Al-
though thousands of fleas have been examined I have unfortunately not yet
been able to study their complete life histories, enough however is known to
shew that extreme precaution is necessary before drawing any conclusions re-
garding the origin of these herpetomonads. It is important to note, that in the
case of both these fleas the infection is acquired by the larvæ, and. this empha-
sizes the futility of conducting experiments with fleas without first studying
their natural flagellates. If this precaution is overlooked, the observer who finds
any of these natural flagellates in fleas fed on mammalian blood, is very apt to
conclude that they represent the extra-corporeal life cycle of a flagellate ingested
by the fleas. Now, throughout all the flea experiments noted above, not a
single observer records the examination of larval fleas, and I believe that, had
this precaution been taken, there would to-day have been no doubt as to the
nature of the flagellates found in Ctenocephalus canis and Pulex irritans. The
fact that these parasites were not found in control fleas is of no value, for I have
examined over five hundred larvæ of C. felis, belonging to a particular batch
hatched out from eggs laid by one lot of fleas, with negative results, while in
another batch of the same larvæ quite a number were found to be infected ;
the number of infected larvæ must depend on the number of infected fleas. The
larvæ of both the fleas I have studied, invariably contain digested blood which
is passed out by the adult insects in their excreta, and this at once explains how
the larvæ become infected with these herpetomonads.
I have therefore no doubt that Basile, Sangiorgi, Alvares, and Pereira de
Silva, have been dealing with one or more species of natural herpetomonas of
fleas, and that these parasites have been mistaken for the developmental forms
*I wi sh t o t ake t hi s opport uni t y of t hanki ng t he Hon. N. Charl es Rot hschi l d for i dent i fyi ng t hi s fl ea.
12
of the parasites of Kala Azar. That this is the case, is clearly shown by
Marzocchi's observations in Piedmont where canine Kala Azar is unknown, for
he states that he has found a flagellate in fleas taken from dogs not infected
with Kala Azar. It is obvious then that the dog flea in Italy contains a natur-
al flagellate, and it is most probable that P. irritans will be found to be infected.
with Crithidia pulicis. From what I have stated above it should be borne in
mind that any flea may contain two distinct flagellates, and it is to be hoped
that those who have the opportunity of studying these forms will not be satisfied
by merely recording parts of their life histories, but will describe them in detail.
In the past this has only been done in a few instances, and as a result, the life
histories of most of these insect flagellates have been confused ; only certain
stages, those most easily obtained, have been recorded, and. new generic names
such as L eptomonas and Leptotrypanosoma have been unnecessarily given them.
I will have more to say on this subject on another occasion. Lastly, those who
are endeavouring to solve these complicated problems in Tropical Medicine, have
been led astray by such unwarranted statements as have recently found their
way into the International Catalogue of Scientific Literature ; I refer to
Woodcock's remarks there and elsewhere, that flagellates found in blood-sucking
insects belong to the extra-corporeal life histories of vertebrate trypanosomes.
The flagellates of blood-sucking insects constitute one of the most serious pitfalls
in connection with this work, and I cannot too strongly urge upon those who
may encounter them in their feeding experiments, to exercise the utmost precau-
tion in accepting Woodcock's statements. I have no doubt whatever that those
flagellates parasitic in the vast majority of blood-sucking insects have no con-
nection with any blood parasite.
It is next necessary to shortly refer to Franchini's recent experiments with
anopheles mosquitoes. This observer believes he has solved the problem of the
method of transmission of infantile Kala Azar : it is however somewhat curious
that no mention is made of the writer's feeding experiments with Neocellia
stephensi. Franchini's experiments were conducted under extremely artificial
conditions, for his mosquitoes were fed on blood containing the cultural forms of
the parasite, and they are in no way comparable with those of the writer carried
out in Madras. In Franchini's experiments there is no certain proof that the
parasite of infantile Kala Azar will pass from its pre-flagellate to its flagellate
stage in the mosquitoes, for the parasites were already flagellates. If it can be
shown, that in this particular anopheles in Italy the parasite will develop
from its pre-flagellate to its flagellate stage, it will then be possible to consider
Franchini's hypothesis as proved, but until this decisive proof is forthcoming it
must surely be left sub judice. In my experiments with Neocellia stephensi
in Madras, I was unable to observe any changes towards the development of the