NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
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filament as seen in the pre-flagellate stage of the parasite ? There can be no
doubt that it is directly connected with the development of the flagellum, but
the view that it represents the root of this organ does not explain its true
nature. It is at present generally believed that the rhizoplast or root of the
flagellum is in some way connected. to the blepharoplast or so-called kineto
nucleus, and. that the flagellum arises de novo from the blepharoplast. As a
result of my studies I believe, on the contrary, that the flagellum is of ecto-
plasmic origin, and that it grows from the pink band or filament present in the
pre-flagellate stage and that this structure is a myoneme. It is well known that
many trypanosomes have myonemes, or contractile bands, and I have seen them
well displayed in Herpetomonas muscœ domesticœ ; in most other herpetomonads,
however, they are so delicate that they cannot as a rule be seen. In many of the
forms I have studied, the pink filament may not only be seen in one part of the
parasite but can be found stretching right across it. In smears made from the
spleen of a case of Bala Azar, or from the blood of an Oriental Sore, I have fre-
quently seen parasites which have ruptured, the protoplasm and nucleus having
been extruded, and the ectoplasm then stains pink, and a well developed band can
be seen stretching across the cell ; very often the bleparoplast remains, and is
then seen lying at right angles to the pink filament. Wenyon depicts this
appearance (ref. fig. 28, plate 12) but he considers it to be an abnormal form.
In support of the view that they are merely ruptured parasites, from which the
protoplasm and nuclei have been extruded, it is only necessary to state that
lying beside many of them there were the nuclei and protoplasmic debris.
The view that the flagellum grows from a myoneme, and is therefore of
ectoplasmic origin, can readily be demonstrated in these parasites by studying
its early development, whereas it has never been seen to develop out of the
blepharoplast ; further I have been unable to find it directly connected with
this rod- shaped structure. I hope on another occasion to return to this subject
and to depict the several appearances in greater detail.
From a reference to figures 12, 13, 14 and 15, it will be seen that the
nucleus may divide first, or on the other hand the blepharoplast, and that the
flagellum may begin to be formed before the parasite itself has divided. These
single enlarged parasites may measure from 7 µ to 8 µ i n l e n g t h , a n d f r o m 5
µ t o
6 µ i n b r e a d t h . A t t h i s s t a g e t h e s e f o r m s m a y e i t h e r d i v i d e a n d t h e n f l a g e l l a t e ,
the two flagella developing side by side, or they may increase in size, and after
the nuclei and blepharoplast have divided, the parasite may itself split into two.
The flagella become extruded. soon after the parasites have separated. When
division of the parasites is about to take place, an indentation forms at one
end, and the line of division passes down, and the parasites may only remain
attached by their flagella, or by both poles. Whatever type of development
22
takes place, the same result is produced, namely, two oval flagellates lying side
by side.
Some of these single parasites, instead of at once dividing, elongate and
result in the formation of spindle-shaped cells ; this appearance is seen in
figures 20 and 26. The spindle-shaped flagellates themselves begin to divide,
and they can then be seen in pairs in all stages of divisions as depicted in
figures 16, 17, 24, 25, 26, 27, 28 and 30.
The second type of multiplication, which is very characteristic of the de-
velopment in the bug, results in the formation of rosettes of from four to more
flagellates. A parasite about to undergo this change, instead of at once
dividing, enlarges, and becomes globular in shape, its nucleus and blepharoplast
now divide, and these in turn divide again, and in this way there may be eight
nuclei and eight blepharoplasts. It is not uncommon to find that either the
nuclei have divided more rapidly than the blepharoplasts, or vice versá. At
the same time the flagella may be seen in all stages of growth, or there may be
only one flagellum. Figures 33 and 41 shew these changes and such cells
may measure from 10 µ to 12 µ in length, and. from 10 µ to 11 in breadth.
As soon as the flagella have grown out, the parasites begin to separate and
break away, figure 29.
All these developmental changes take place both in Cimex rotundatus and in
Cimex lectularius from the third to the fifth day after the last feed of infected
blood. The formation of the spindle-shaped or elongated flagellates may then
take place in one of the following ways :—
(1) A young growing parasite may flagellate, and without dividing,
elongate and become spindle-shaped, but this is not the usual
method of development.
(2) A growing parasite, instead of at once flagellating, divides, and. the
resulting pair become elongated, and they may in turn divide
again ; or
(3) A parasite instead of flagellating or dividing, enlarges, and the nuc-
leus and blepharoplast divide up into eight or more, the flagella
then grow out and the cell splits up into many elongated flagel- -
lates.
It should be noticed that many of the oval or spindle-shaped flagellates
themselves divide up regularly or irregularly into several shorter or longer forms
depending on the size of the parent cell. Figures 31, 32, 36, 37, 38, 39 and 40
depict these changes. The resulting flagellates, whether they be long or short,
are very characteristic. Their protoplasm stains dark blue and appears to be
granular and vacuolated ; their nuclei, oval in shape, are usually situated about
the centre of their bodies, they stain pink and contain many chromatic rods.
23
Their blepharoplasts may be rod-shaped or circular, and always lie closer to the
nuclei than to the anterior ends of the parasites. Their flagella stain deep pink
and consist of many delicate filaments adhering together, and arise from a point
a little anterior to the blepharoplasts.
We have now seen that by the fifth day the parasites, have grown and mul-
tiplied, so that in place of the original pre-flagellate stage ingested in leucocytes,
the parasites have not only become free, bat they have increased in numbers
and are now long actively moving flagellates.
The spindle-shaped or elongated flagellates, which predominate from the
f i f t h d a y o n w a r d s , n o w b e g i n t o d i v i d e u p a n d t h e r o s e t t e s a r e s e e n a s m a s s e s
with their flagella intertwining and directed inwards. In many of these
spindle-shaped forms a process of unequal division was observed, such as was
first described by Leishman from cultures in vitro. Parasites, which appear to
be dividing normally into two more or less equal flagellates, exhibit this curious
unequal division resulting in the splitting off of spirilla-like daughter cells.
These may contain neither nuclei or blepharoplasts and frequently they only
have one chromatin mass. I do not propose describing these parasites further,
beyond remarking that several were seen both in rotundatus and lectularus ;
I will describe them more in detail on another occasion and will then explain
what I consider to be their true nature.
In several of the bugs, particularly in lectularius, many large rosettes such
as are depicted in figures 45 and 46 were seen, and the bugs contained a large
number of flagellates, such as are shewn in figures 42 and 43. These mature
flagellates measure from 12 to 20 µ in length and from 4 µ to 5 µ in breadth,
their nuclei are nearly always oval in shape and lie about the centre of their
bodies ; the blepharoplasts may be rod-shaped or circular. The posterior ends
of the parasites are pointed, while their anterior ends are rounded and their
flagella measure about 21 µ in length. In my second report I stated that I was
then unable to observe equal division taking place in these long flagellates, but
I am inclined now to think that they may divide further in this way, but this
division was not seen in many instances. In the rosette shewn in figure 46
there is some evidence that the parasites were dividing up in this manner.
By the sixth and seventh days the parasites were nearly all elongated
flagellates, either free or massed together in rosettes. It should here be noted
that several groups of flagellates were seen in the films, attached to cells by
their flagella. Figure 40 represents such a group, and in a fresh preparation
this was very clearly demonstrated ; it is exactly similar to what takes place in
the case of all insect herpetomonads as they approach their post-flagellate stage.
Figure 41 requires special mention. Many of the spindle-shaped flagellates
exhibited a curious appearance at their posterior ends as shewn in this figure ;
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a part of the protoplasm appeared to be breaking off, and in some it was seen
as a mere tag. It was at first thought that this appearance was merely the
result of the pointed end becoming twisted, but it was seen in many of these
dividing forms, and I am at present inclined to think it is a portion of residual
protoplasm being thrown off.
The Post-flagellate Stage.
The whole interest in the life history of this parasite, centres round the
final stages in its development, the nature of which was predicted in 1907 ; it
is true some observers claim to have seen this stage in cultures in the test tube,
I however fail to understand how this is posssible. Franchini records having
found cystic forms in old cultures of the parasite of infantile Kala Azar, and in
my opinion he rightly regards these as degenerating parasites. It is interesting
to note that in several of the bugs which had a second feed on the other case
of Kala Azar recorded above, such round degenerating forms were seen ; these
had undoubtedly once been flagellates and the fresh blood had caused them to
degenerate, and assume a rounded form. In the recent number of the Kala
Azar bulletin on page 49, there is a note by the writer to the effect that
Dr. Row has succeeded in tracing the final stages of the parasite of Oriental
sore of Cambay in cultures in vitro. It remains to be seen how the morpho-
logy of the forms discovered by Dr. Row, correspond with those now to be
described. It should be noted that, if in the bug fresh blood causes the flagel-
lates to soon round up and degenerate, it is only natural to expect that exactly
the same would take place when the culture containing flagellates is transferred
to fresh medium, which presumably contains blood. The extreme sensitiveness
of these flagellates of the parasite of Kala Azar to human or monkey's blood
makes me doubt whether these rounded forms, found in old cultures of the
parasites of Kala Azar, and Oriental sore, are anything more than degenerating
forms.
The post-flagellate stage in the bug begins on about the eighth day, and
appears to be completed by the twelfth. It was noticed that the mature
f l a g e l - l a t e s o n t h e s e v e n t h , e i g h t h a n d n i n t h d a y a l t e r e d i n a p p e a r a n c e , t h e b l u e
staining protoplasm, which is characteristic of all the earlier forms, had now
changed, and the flagellates were a light pink ; further many were now seen
with short flagella as is depicted in figure 50. Several other important struc-
tural details left no doubt that the long looked for post-flagellate stage was at
last found. Figures 47, 48 and 49 represent the earliest changes towards the
final rounding up of the parasite. These flagellates are more or less oval in
shape, and measure from 9 µ to 12 µ i n l e n g t h a n d f r o m 5
µ t o 6
µ i n b r e a d t h ;
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their protoplasm stains light pink, it is almost homogenous, and. is surrounded
by a well marked pink staining periplast. The other outlines of their nuclei
are obscured and. they are full of small pink rods ; their blepharoplasts stain as
before. Most of these forms were undergoing division as evidenced by their having
two nuclei, two blepharoplasts, and two flagella. Any one who has seen this stage
and compared it with the younger growing forms depicted in figures 25, 26, 27,
and 28, could not confuse the two. The young parasites stain blue and their
nuclei are well defined. It may be thought that these rounding up forms are
degenerating flagellates ; in order to disprove this view, bugs were examined at
this stage in the fresh condition, and it was found that these forms were
extremely active and that they were attached to the intestinal wall by their
flagellar ends. When the gut was ruptured, they were seen lashing about while
still attached. I have never before seen the flagellate stage of the parasite
exhibiting such active movements, those I have previously seen only shewed
slow movements ; this was obviously due to the fact that the bugs had fed
again.
Like the post-flagellate forms of all other herpetomonads, the parasite of
Kala Azar then begins this stage by attaching itself to the intestinal wall, and
there it slowly rounds up and at the same time divides again. On the comple-
tion of this final division, smaller forms are produced, and these again divide,
resulting in the final stage which is depicted in figures 67 to 73. Several of
these rounding up parasites had long flagella, figure 61, while the majority only
possessed short stumpy ones and many merely the remains of flagella projecting
as faintly staining filaments ; figures 51 to 66 chew these appearances. Here
again it may be said that such forms as are depicted in figures 52, 53, 54, 55,
60 and 62 are young forms about to flagellate. I have carefully described that
stage of the parasite which proceeds the extrusion of the flagellum, and have
drawn attention to its appearance. The only way the round post-flagellate
resembles it, is in its shape, otherwise they are totally different. The smallest
one I have seen is depicted in figure 67 and the majority are larger than the
corresponding pre-flagellate stage. They have ill-defined nuclei which contain
many granules ; the blepharoplast, which is also present, is not much altered.
These forms stain well and have a distinct pink-staining periplast. In none
of these parasites were any of the appearances seen which could possibly
suggest that they were pre-flagellates just about to enlarge and flagellate. It is
important to note that when they appeared in the bugs, they were the predomi-
nating forms, whereas just a few days previously, flagellates were the only
parasites to be seen.
It is necessary now to shortly consider how the parasite finds its way back
to man when the bug feeds again. The life history, I have described above,
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takes place in the midgut of the bug, and throughout its development it never
leaves it. I have so far searched in vain in the salivary glands of the bug for
the post-flagellates ; this stage appears to be completed in the midgut of the-
bug. How then can they be injected into man ? This I believe takes place
by regurgitation, as there is no structure in the upper part of the alimentary
tract of the bug which could prevent this happening. The bug has no thicken-
ed proventriculus which could act as a constrictor and prevent any of the-
contents of the midgut from passing up the œsophagus into the pumping
organ in its head. Further, the bug has the curious habit of returning to feed
after it is replete with blood ; and I have seen one insert its proboscis, no less
than six times, before it was satisfied ; all the stages of the bug behave in this.
way. On examining the first nymphal instar, after it has fully fed, under a
low power lens it will be seen that the midgut is pressed right up into its
prothorax, and that the bright red blood can be readily seen in its head. If
such a bug contained the post-flagellates ready to be injected, they would tend.
to be loosened by the peristaltic movements which follow the ingestion of blood,
and if the bug returned again to feed, there is no difficulty in understanding
how they may be regurgitated into the wound. I do not intend however to try
and explain further this part of the problem, for I hope in my next experiments.
to endeavour to solve it once and for all.
There is one point, which is specially brought out in these experiments, and.
that is, that the parasite will develop as readily in Cimex lectularius as in Cimex
rotundatus ; I have long wished to utilise this bug, but up to the present I
have not succeeded in doing so. It was further noticed, that the development
in the European species was, if anything, more marked than in its oriental ally,
this may only be due to the fact that the digestion of the blood in it takes place
more slowly, thus giving the parasites the maximum amount of nourishment.
As a result of the experiments with Cimex lectularius I venture to predict that
the closely related parasite of the Mediterranean littoral will also undergo its.
development in this bug. The experiments which have been carried out with
this insect have, up to the present, been conducted under artificial conditions,
and they in no way simulate those described above ; the search for parasites in
bugs caught at large is a waste of time, for the reason that though the bug may
have fed on a suitable case it may have fed too often with the result that all
the flagellates may have been destroyed.
I find myself in entire agreement with Gabbi who believes that Infantile
Kala Azar is not of canine origin, and consider that those who believe the
contrary should bring forward much more convincing proof than has yet been
produced.
In order that any weight may be attached to feeding experiments, both
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with fleas and mosquitoes, it is very necessary for the observer to shew that he is
aware of the many pitfalls which may be encountered in such work, and to take
every precaution to exclude natural parasites of the insects. Further, I would
emphasize this point, that before it is possible to accept a statement that the
parasite of Kala Azar will multiply in any insect, it is of the first importance
for the observer to shew that it actually develops from its pre-flagellate stage.
This has up to the present not been done. Lastly I may say that I attribute
all my success in this work to the valuable knowledge gained by studying
insect herpetomonads ; the working out of their life histories is almost of equal
importance to the study of the extra-corporeal life history of the parasite of
Kala Azar. I have studiously refrained from recording only parts of the
developmental stages of these harmless flagellates, for owing to their great
pleomorphism they exhibit such varying forms, that as a result, they have been
much confused in the past and still less understood. They form, as a matter of
fact, a much more homogenous group than do the trypanosoma . It is more
particularly those forms which occur in blood-sucking insects which. deserve
the most careful attention of Protozoologists and students of tropical medicine.
The fact that one, and possibly more than one, of these human herpetomonads is
a true insect parasite in that it undergoes the most important part of its life
history in its invertebrate host, should at once arrest our attention, for it is no
imaginary hypothesis to think that any one of these, at present, harmless para-
sites, may become pathogenic to man or the domestic animals. The fact that
the herpetomonas of anopheles mosquitoes may undergo its post-flagellate stage
in the midgut of the insect suggests that at least this parasite is varying its life
history.
The behaviour of the Parasite in Conorrhinus rubrofaciastus, de Geer.
Mesnil, commenting on my discovery of the development of the parasite in
Cimex rotundatus, has expressed the view that it does not follow, that, because
the parasite flagellates in this bug, it will prove to be the true invertebrate
host ; the development may merely take place, owing to the fact that the bug
ingests a large quantity of blood, and that its midgut then simulates a culture
tube. Wenyon has further elaborated this view, and in his report on Oriental
Sore in Bagdad, when referring to the development of the parasite he has
observed in the bug, he makes the following statement : " These observations
are very similar to those made by Patton on the development of the parasite of
Kala Azar in Cimex rotundatus. If the parasite of the sore can develop into a
flagellate in the stomach of an insect, not its true host, then it would be expect-
ed. that the very similar parasite of Kala Azar would develop in the same
manner. Such experiments and. results only shew that it is unsafe to draw the E
28
conclusion that an insect showing such developmental stages in its gut is the
true carrier of disease, for there is no question of the possibility of the bed-bug
being the carrier of the sore in Bagdad. This development is then merely a
partial imitation of what would actually take place in the true intermediate
host." In a still more recent note, Wenyon states that he " has been driven to
the conclusion that the parasite of Oriental Sore develops in the intestine of
the bed-bug in just the same way as in artificial cultures in various blood-con-
taining media. In connection with Patton's discovery of the development of
Leishmania donovani in the bed-bug Mesnil has made the same suggestion, so
that such a development does not necessarily mean that the insect in question
is the natural host of the parasite. It is highly probable that the parasites of
the group Leishmania will develop to a certain extent in the intestines of
many insects, provided that they find there a suitable pabulum rich in blood.
The development I have described of Leishmania tropica in Stegomyia fasciata,
and that of Leishmania donovani in Anopheles by. Professor Franchini, may
be of this nature, and only, so to speak, accidental, owing to the rich nutrient
fluid taken up with the parasites by these blood-sucking flies." In connection
with these statements it is first necessary to point out that the parasite of
Indian Kala Azar will not flagellate in Culex fatigans, Neocellia stephensi,
Stegomyia sugens, Ornithodorus savignyi, Pediculus capitis, and Pediculus vesti-
menti, in spite of the fact that these insects, particularly the mosquitoes and the
tick, suck up large quantities of blood. Precisely the same was noted in the case
of the parasite of Cambay Boil, it will not flagellate in Stegomyia. Further I very
much doubt whether Wenyon actually observed the flagellate stage of the para-
site of Bagdad Boil in Stegomyia fasciata. He admits having found a herpe-
tomonas in the larva of this insect and it is inconceivable to think that if it is
present in this stage it will not be present in the nymph or the adult insect ; it
is very probable that when the female mosquitoes fed on the boils the flagellates
disappeared in much the same way as did those in Culex fatigans noted above,
and that they only persisted in a few. The conclusive proof that the flagellates
observed by Wenyon in Stegomyia fasciata were the developmental forms of
the parasite they had ingested, is unfortunately wanting, for although he figures
the early stages they are not sufficiently convincing ; if the parasite regularly
develops in this mosquito there would be no difficulty in following all the stages
in its growth, from the unchanged parasite in a cell, up to the formation and
extrusion of the flagellum, such as I have repeatedly observed in the bug. In
the local Stegomyia of Cambay I failed to observe any development, and this
mosquito never contained a natural herpetomonas, thus my failure to find
flagellates or round forms.
There is therefore at present no proof that either the parasite of Kala Azar,