NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
TABLE III .—Shewing in tabular form the results of feeding experiments.
Litter.
Number
of
Dog.
Species of tick.
Stage of tick.
Date on which
ticks were added
to cage.
Date on which
infection was
first noticed.
Incuba-
tion
period
in days.
REMARKS.
No. 1
1 0 0
R. sanguineus . Larvæ
. . 25th Oct. '06 .
......
. . .
No infection on 7th November 1906 or 13 days
after being bitten.
No. 1
1 0 1
Do.
.
Do.
. . 25th Oct. '06 .
......
. . .
No infection on 8th November 1906 or 14 days after
being bitten.
No. 1
102
Do.
.
Do.
. . 25th Oct. '06 .
......
. . .
No infection on 10th November 1906 or 16 days after
being bitten.
No. 1
103
Do.
.
Do.
. . 25th Oct. '06 .
. . . . . .
. . .
No infection on 8th November 1906 or 12 days after
being bitten.
No. 1
104
Do.
. Do.
. . 25th Oct. '06 .
......
...
No infection on 9th November 1906 or 15 days after
being bitten.
No. 2
105
Do.
. Nymphs
.
. 13th Nov. '06 . 17th Nov. '06 .
4
Mild attack. Piroplasma in blood.
No. 2
106
R. bursa
. Larvæ, nymphs
and adults.
25th Oct. '06 .
......
...
No infection resulted. Destroyed after some weeks.
No. 2
107
R. sanguineus . Nymphs
.
. 13th Nov. '06 . 16th Nov. '06 .
3
Severe attack. Piroplasma in blood.
No. 2
108
R. bursa
. Laræ, nymphs
and adults.
25th Oct. '06 .
. . . . . .
. . .
No infection resulted. Destroyed after some weeks.
No. 3
109
R. sanguineus . Larvæ
.
. 25th Oct. '06 .
......
. . .
Do.
. Nymphs
.
.
4th Nov. '06 . 12th Nov. '05 .
8 ? Acute attack. Piroplasma in blood.
No. 3
110
Do.
. Larvæ
. . 25th Oct.
' 0 6
.
. . . . . .
. . .
Do.
. Nymphs ? .
. 4th Nov. '06 . 12th Nov. '06 .
8 ? Died 15th November 1906. Piroplasma in blood.
No. 4
1 1 1
Do.
.
Larvæ .
Oct. '06 .
......
...
Nymphs
.
. 4th Nov. '06 .
8th Nov. '06 .
4 ? Died. Piroplasma in blood.
No. 4
112
Do.
.
Larvæ
. . 27th Oct. '06 .
. . . . .
...
Nymphs
. .
4th Nov. '06 .
8th Nov. '06 .
4 ? Died. Piroplasma in blood.
No. 4
113
Control .
. ......
......
. . . . . .
...
Remained free from infection until destroyed on 16th
November 1906.
52
TABLE III.—Shewing in tàbular form the results of feeding experiments— contd.
Litter.
Number
of
Dog.
Species of tick.
Stage of tick.
Date on which
ticks were added
to cage.
Date on which
infection was
first noticed.
Incuba-
ti on
perio d
in days.
REMARKS.
No. 6
118
R. bursa .
. Nymphs and adults
......
. . . . . .
. . .
No infection resulted.
No. 6
119
Do.
.
.
Do.
......
. . . . . .
. . .
Ditto.
No. 7
120
R. sanguineus
Larvæ
.
. 28th Oct. '06 .
......
...
Nymphs
.
.
4th Nov. '06 .
9th Nov. '06 .
5 ? Died. Piroplasma in blood.
No. 8
121
Do.
.
Do.
.
.
13th Nov. '06 . 17th Nov. '06 .
4
Died. Piroplasma in blood.
No. 8
122
Do.
.
Nymphs ? .
.
13th Nov. '06 .
. . . . . .
. . .
Adults
.
.
22nd Nov. '06 . 28th Nov. '06 .
6 ? Died. Piroplasma in blood.
No. 10
126
Control
. .
. . . . . .
......
. . . . . .
. . .
Kept from 27th October 1906. Destroyed after some
weeks. No piroplasma.
No. II
127
R. sanguineus . Adults
.
29th Nov. '06 .
4th Dec. '06 .
5
Recovered. Piroplasma in blood.
No. II
128
Control .
.
. . . . . .
......
......
. . .
No infection resulted.
No. II
129
R. sanguineus . Adults
. . 29th Nov. '06 . 4th Dec. '06 .
5
Piroplasma in blood.
No. II
130
Control .
.
. . . . . .
. . . . . .
......
. . .
No infection resulted.
No. 12
131
R. sanguineus . Adults
. . 11th Dec. '06 . 21st Dec. '06 .
10
Piroplasma in blood.
No. 12
132
Do.
.
Do.
. . 11th Dec. '06 . 21st Dec. '06 .
10
Ditto.
No. 14
135
Do.
.
Do.
. . 10th Dec. '06 . 18th Dec. '06 .
8
Dying state when first observed. Piroplasma.
No. 14
136
Do.
.
Do.
.
. 10th Dec. '06
18th Dec. '06 .
8
Blood swarming with piroplasma when first
observed.
No. 15
137
Control .
.
. . . . . .
. . . . . .
. . . . . .
...
From 1st December 1906. Remained throughout
experiments free from infection.
No. 15
138
Do.
.
.
......
......
......
...
Ditto
ditto
ditto.
No. 15
139
R. sanguineus . Adults
.
. 10th Dec. '06. . 18th Dec. '06 .
8
Swarming with piroplasma when first observed.
No. 15
140
Do.
.
Do.
.
. 10th Dec. '06 . 18th Dec. '06 .
8
Ditto
ditto
ditto.
53
55
V.
DEVELOPMENTAL CYCLE OF PIROPLASMA CANIS
IN THE TICK.
Trustworthy observations upon developmental stages of piroplasma in the
tick prior to those of Koch (188 and 189) do not, so far as I am aware, exist.
Koch notes that in the gut of certain ticks R. australis, R. evertsi and H. ægyp-
tium, Piroplasma bigeminum increases in size and develops long ray-like pro-
cesses. Later on certain of these star-shaped forms are seen associated in
couples, an association which Koch considers may be a kind of conjugation.
Eventually the processes are retracted and the bodies assume a globular shape.
Koch also describes and figures some other stages, notably masses of young
amœboid parasites and, in the egg, certain large club-shaped forms four times
the size of piroplasma in the blood.
A similar formation of star-shaped forms is also noted by Koch in the case
of the minute parasite of East Coast fever, in this case only in the tick R. austra-
lis. * The earlier portion of this development is described by Kleine as taking
place in vitro. My own observations, however, confirm this only in a very modi-
fied way, it being very doubtful whether the formation of processes such as those
described by Koch and Kleine is an essential part of development.
Though these facts are a considerable advance upon previous knowledge
they do not enable one to assign any particular cycle of development to piro-
plasma; and the relation of the club-shaped bodies to the other stages is quite
indefinite.
In a preliminary note (185) I described in addition to those just mentioned
certain other developmental forms of piroplasma and spewed that development
eventually resulted in swarms of bodies somewhat resembling piroplasma as
seen in the blood and that these were congregated in the salivary gland of
nymphs of the second generation.
Unfortunately at the time of writing this note I was unaware that infection
could be transmitted by R. sanguineus not only hereditarily, but directly from
the nymph to the adult, and that in some cases I had been dealing with
nymphs shewing both forms of infection. A realisation of this fact made it
apparent that the developmental cycle was in reality much simpler than I had
supposed, and in the present memoir I am able to give with some degree of
certainty at least the outlines of development of the parasite, and to indicate
* Vide, however, Lounsbury's and Theiler's experiments on the transmission of this parasite.
56
the main facts regarding the mechanism by which infection is transmitted both
in hereditary and in stage to stage infection.
The technique used has been based mainly on a knowledge of the anatomy
of the tick. The principal features of this have been given by me in a previous
publication and some further points which have an importance in relation to
the development of piroplasma have already been mentioned in this Memoir
(latter portion of Part III).
Gorged females are examined by partial dissection and the removal of por-
tions of the gut or of the ovary and oviduct for examination fresh or after the
preparation of films. In fresh preparations the club-shaped forms are generally
to be found without difficulty, once their general appearance is known, in or
about the ovary and oviduct, especially if a low power (1/6 objective) is used for
their detection.
Developmental forms in the gut of the adult are usually more difficult to
obtain. It is probable that this portion of the development takes place rather
quickly and that the parasites have at the period of full engorgement already
left many of the diverticula. The best results have been obtained by removing
partially fed adults at a time when, by a series of examinations, it is known that
the dog has passed through a period of intense infection immediately before.
Preparations shewing the tissue of the developing adult have been obtained
by cutting off with a sharp scalpel the anterior third of the gorged nymph at
various periods after it has dropped from the host, and after turning out the
embryonic tissue making squash preparations in the usual way.
Such a procedure enables one to avoid the gut contents which remain
almost entirely in the discarded posterior portion of the nymph.
FORMATION OF THE CLUB-SHAPED BODIES.
In preparations of the gut of ticks fed on dogs whose blood has contained
numerous parasites the early stages of development of the parasite are seen.
This development occurs in parasites not distinguishable from ordinary free
forms,* which increase considerably in size becoming globular bodies 4 to 5 µ
in diameter. In the largest forms an achromatic line appears which divides
the parasite into two nearly equal segments ; only one of the segments contains
chromatin, an appearance which was wrongly interpreted by me in my earlier
note as being of the nature of conjugation (fig. 4, 3). The two portions
marked off by the achromatic line eventually separate from one another except
at one point, and the portion devoid of chromatin swings round to form the
tail-piece of the club-shaped body, the rest of the parasite becoming the front
* Like myself, Nuttall and Graham Smith have failed to detect any forms of the parasite which there
seems any reason to regard as "gametes."
57
half of this structure (fig. 4, No. 4). After the separation of the tail-piece there
are often irregular jagged processes and the parasite appears as shewn in P1. 1,
fig. 56, but later all inequalities become smoothed off and a perfect but imma-
ture club-shaped body results. Shortly after being formed the club-shaped
bodies leave the gut.
FIG. 4.
Shewing origin of single and double club-shaped bodies.
I — 5 . F o r m a t i o n o f s i n g l e c l u b - s h a p e d b o d i e s .
I'—4.' Formation of double club-shaped bodies.
Fig. 1. Parasite undergoing enlargement.
Fig. 2. Enlarged parasite before the appearance of achromatic line.
Fig. 3. Parasite with achromatic line marking off future tail-piece.
Fig. 4. Separation of tail-piece.
Fig. 5. Fully formed club-shaped body.
Fig. 1'. Parasite in early stages of fission enlarged.
Fig. 2'. Round body with complicated achromatic lines which indicate the line of fission producing
two bodies as shewn in fig. 3'.
Fig. 3'. Double body.
Fig. 4'. Double body in which each half has developed into a club-shaped body.
The area of chromatin indicated by the single line is the representative of the extension seen in
piroplasma in the blood.
If the formation of long processes really takes place as described by Koch
and Kleine it must be in the early stages during which the parasite is increas-
ing in size, prior to the separation of the tail-piece. Parasites exhibiting some
such appearance certainly do occur, but as a rule some irregularity of outline
only is noticed, and in many cases development seems to take place without
any processes at all being formed. Whether the forms figured in Pl. 1,
fig. 48, which resemble certain forms figured by Koch, have any particular
significance I am unable to say.
As a rule parasites shewing development are found lying by themselves,
either in the gut contents, or, as it would sometimes appear, embedded in the
protoplasm of a cell of the gut. At other times large groups of forms are
58
encountered as shown n Pl. I, fig. 53. The occurrence of these large groups at
first led me to suppose that in the gut the parasite underwent fission, but I have
since come to the conclusion that such groups are merely the outcome of
development of a cluster of original parasites. In support of such a view is
the fact that double forms and groups of four are also common. Even club-
shaped bodies are frequently seen in pairs or in a group of four closely situated
forms (Pl. II, figs. 13 and 12).
In addition to the ordinary forms there are certain very peculiar bodies
possessing a double chromatin mass and shewing achromatic lines (Pl. I, fig. 52).
Koch has figured a body of this nature ( vide his Pl. II, fig. 24) and con-
siders it a stage in development of the parasite. They appear to be merely
instances where a parasite on the point of division has been taken into
the gut and has proceeded to develop. The achromatic lines have the same
significance as in the single forms, but indicate in this case the lines of cleavage
in the formation of two club-shaped bodies, which at this stage are short stumpy
forms as shewn in fig. 4.31. The results of division of these forms are usually
shuttle-cock-shaped bodies (Pl. 1, figs. 54 and 55). They are often seen still
connected by their bases so as to form double forms, but many are seen lying
free.
The double forms certainly at first sight suggest some kind of conjugation
and it is almost certain they are the forms described by Koch, though they
do not possess the sharp ray-like processes described by this author. Each
parasite, however, develops into a club-shaped body and it is evident that the
single forms are the result of separation of the double forms and not vice versâ.
The method of formation of these bodies and of the ordinary club-shaped
forms is diagrammatically represented in the accompanying figure.
In my preliminary note I described club-shaped bodies as occurring in
hereditarily infected nymphs and I was puzzled to account for the persistence or
recurrence of these forms until it became clear that they arose merely from a
reinfection of the nymphs from dogs which had become infected as a result of
the bites of other nymphs of the same brood. This complication it is easy to
understand, since there would be a considerable interval between the time of
attachment of the first and last nymphs of a batch, an interval more than suffi-
cient to bridge over the short incubation period which was present in these
experiments.
As the result of feeding nymphs on infected dogs it was discovered that
the same development of the parasite takes place in the gut of this stage as in
the gut of the adult and, as in the latter case, results in the formation of club-
shaped bodies, each club-shaped body being the direct representative of an original
parasite in the blood. Since these club-shaped bodies are found to go through
59
development and, as described later, to form sporozoits in the salivary glands of
the adult, it is practically certain, though not yet proved by actual experiment,
that in R. sanguineus infection taken in by the nymph can be transmitted by the
adult.
Whether any sexual process is involved in the formation of the club-shaped
bodies cannot yet be definitely stated; but there are sometimes seen parasites in
which the chromatin is arranged in the form of granules scattered over the sur-
face, and in one instance a very significant appearance was seen where a smaller
pear-shaped form had apparently separated itself from such a body.
The method of development in the tick certainly suggests a sexual cycle,
and it is immediately prior to the formation of the club-shaped bodies that one
would expect the junction of the male and female elements to occur. As just
stated, the double forms are not conjugation forms and the analogy of develop-
ment of piroplasma with that of the malarial parasite would lead one to suppose
that the male element is small, and in such a situation as the gut of the tick
likely to be easily overlooked. The absence of any definite observations regard-
ing fertilisation is not therefore sufficient at this stage to negative the view that
the cycle of development in the tick is a sexual one.
Club-shaped bodies.
These bodies whose origin we have just described are found in the gu
of the adult tick fed on infected dogs, and in still greater numbers about the
ovaries and oviducts, and in the ova. In nymphs fed on infected dogs they are
found in the gut and in the tissues.
There is no appreciable difference between the bodies as seen in the adult
and the nymph, and their origin in both cases is obviously from a parasite taken
into the gut of either of these forms.
They are most easily detected in fresh preparations and are visible even
under a 1/6 dry objective. Under a high power two types are seen.
(a) Rather rigid thorn-like bodies resembling on a very small scale certain
gregarine trophozoits. This resemblance is largely due to the presence of a
peculiar disc-like structure armed with cusps carried at the anterior extremity.
They are sometimes motionless, but usually exhibit rather slow movements, es-
pecially a side to side flap-like action of the tail portion. The protoplasm is
transparent, slightly refractile, and free from large granulations, and in it a clear
area (the nucleus) can often be made out.
The disc has the appearance of a boring organ. It carries four or five cusps,
one of which is situated centrally, whilst the others are arranged round the
periphery. Immediately behind this structure the parasite is often constricted so
that a kind of neck is formed.
I 2
60
(b) Leech-like forms more club-shaped than those just described and
executing very active movements. They possess a swollen end which is often
attached to the slide or cover glass and a thin end which is kept in constant
motion like the head of a leech. They also undergo modified amœboid move-
ments, the shapes depicted in P1. II, fig. 2, being characteristic.
These bodies are most numerous as a rule about the oviducts, and a few
can often be found here when not to be detected elsewhere. They also occur in
the ovary and may be seen lying within ova not yet set free in the lumen, and in
more mature ova. By careful search they can also be found in fresh prepara-
tions of the gut.
In stained preparations a greater variety of forms is apparent, but the two
forms mentioned, those with and those without a disc, are distinguishable.
Since intermediate stages are not difficult to find it is probable that both are iden-
tical in nature, the disc bearing forms being the more mature stage.
The protoplasm of the club-shaped forms is finely granular and stains a
light blue with Giemsa stain. It is often homogeneous but denser towards
the periphery; very frequently it is vacuolated. The vacuoles when present are
large irregular non-refractile spaces occurring, as a rule, on either side of the
chromatin mass which most frequently lies in a portion of protoplasm free
from vacuoles. There is sometimes a darkly staining spot at the tail end, and
a small spike described by Koch is sometimes visible. Appearances suggesting
that the parasite has a leaf-like form are common, but may be the result
of preparation of the specimen. A common appearance in the less mature
forms is a constriction about the middle of the parasite due to the method
in which the body is formed.
The chromatin consists in the younger forms of a dense mass with a lightly
staining extension, lying most frequently at right angles to the parasite as it
often does in piroplasma in the blood; at other times the extension is directed
towards the pointed end or even anteriorly, a position which it assumes imme-
diately prior to the formation of the disc. In the mature club-shaped forms there
is as a rule no extension, the chromatin consisting of a single dense mass
surrounded by a pale achromatic area. It is circular or irregular in shape and is
often star-shaped with sharp ray-like processes.
The disc and very often a portion of the neck may stain red like the
chromatin or a dark purple. It shews very often an achromatic line around its
circumference. The first indication of the formation of the disc is the passage
forwards of a portion of the chromatin which is at first attached by a filament to
the main mass but later becomes separated (P1. II, fig. 10). The separated
portion of chromatin then divides and eventually becomes diffused throughout the
anterior end of the parasite. Later on the anterior extremity of the parasite
61
stains a deep purple and the chromatin appears to have disappeared. Many
forms appear to proceed to develop into zygotes even without having possessed a
disc.
Double club-shaped forms are commonly seen. They originate from the
double forms already described, in which case they are attached by the broad
end, or they may occur by fission of certain very stout club-shaped forms in
which case they are generally arranged alongside one another (P1. II, figs. 15
and 17).
As already noted the club-shaped bodies leave the gut and are found in the
ovary and oviducts of the adult or in the tissues of the nymph.
In both situations they undergo changes, becoming converted into globular
or oval bodies having a characteristic appearance which I have for convenience
of description and on account of its analogy with this stage in the malarial
parasite called the "zygote."
The change of the club-shaped body into the zygote can be followed, forms
being seen in which the thicker end of the club-shaped body has become
swollen whilst the tail end is still recognisable (Pl. II, figs. 18 to 21), or in which
the body of the club-shaped form is swollen so as to form a globular mass, but
both the head and tail end are still traceable. In some cases a dark area, the
remains of the disc, is still evident on the periphery of the young zygote.
The Zygote.
The zygotes when first formed are oval or round bodies in which the proto-
plasm stains a dark blue with Giemsa stain and the chromatin of which at first
resembles that of the club-shaped bodies. They generally lie within a cell
though they are frequently seen free, possibly the result of rupture of the cell
in the making of the preparation. In the ovum they lie in the yelk, but in the
nymph they lie embedded in the protoplasm of a cell, two and even three zygotes
sometimes being present in a single cell. In some instances the zygote lies in
a space in the cell, but more usually they are embedded in the protoplasm so
that their outline is seen only on careful examination. In most cases the
young zygote is more or less globular, but not infrequently they seem to have
processes extending into the protoplasm of the host cell. They increase greatly
in size, becoming very large somewhat irregular bodies as much as 25 µ in
diameter. The chromatin, at first identical with that of the club-shaped bodies,
increases greatly in amount and sends out long filaments into the protoplasm.
After a time the chromatin has formed two and later on as many as half a dozen
main masses, and from these extend a plexus of chromatin filaments many of
them ending near the periphery of the parasite n a slightly clubbed extremity.