NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
22
position of these masses in different forms of the parasite, and also refer to other
appearances which I shall discuss later. Fantham* has also observed the
loose area noted by Nuttall and figures a relation of the blepharoplast to this
such as is shown in fig. 1, No. 1. To demonstrate the chromatin I have used
various degrees of colouration of Giemsa's stain and the very intense modifica-
tion of Romanowski's stain described by Wright, in which by careful washing of
intensely stained specimens with weak alcohol very clear results can be obtained.
Variations in the position of the large chromatin mass appear to be due
mainly to the positions which it has occupied at various times after fission.
It lies as a rule somewhat near the periphery so that it may appear from
different points of view on either side or in the centre of the parasite. Again, as
after fission it passes round the periphery, it may be at various levels between
the base and the apex of the pear.
In the multiple forms it is more central in position. In the refractile forms
it is pressed to the periphery and may be flattened or drawn out to a rod shape.
The blepharoplast is often seen as a distinct and very markedly punctiform
body. It is, however, by no means always present. In my specimens it has
generally been associated with changes in the third area of chromatin and with
division of the parasite. (Pl. 1, figs. 32—39 and fig. 1, page 21.)
The loosely packed area described by Nuttall and Graham Smith is almost
invariably present, and its apparent absence is generally a result of imperfect
fixation or staining ; in certain small round forms of infrequent appearance it is,
however, often not visible.
Nuttall and Graham Smith figure this mass as in some cases continuous
with, and in others separate from, the main mass. As a result of close obser-
vation I am convinced that it is rarely, if ever, really separate from the main
chromatin mass, though its main bulk may be so ; as a rule it is obviously con-
tinuous with and appears as an extension of the main mass.
In many preparations the area consists of a fine irregular reticulum of
chromatin threads, in others it appears as an area of homogeneous material
having clearly defined edges from which there may arise one or more fine
chromatin filaments. In some cases the extension clearly consists of two or
three long chromatin filaments, which can be traced almost the full length of
the parasite.
In some cases the filaments are aggregated to form a thick cord, in others
they are more spread out. In the large " early invasion" forms a fan-like arrange-
ment is common. In the round refractile forms the main mass is flattened upon
the periphery with a long straggling projection of loose chromatin usually lying
* Private communication.
23
along the periphery, but sometimes spread as a thin streak over the refractile
centre (Pl. 1, fig. 29).
An arrangement of the chromatin of the same nature is seen in the develop-
mental forms of piroplasma in the tick. In both situations this extended or
filamentous portion of the chromatin first heralds division of the parasite. In
single forms in the blood which are in the early stage of growth the chro-
matin consists of the main mass with a small extended area (fig. 1, No. 1). As
growth proceeds and division approaches, the extended area becomes more
pronounced and spreads further from the main mass. It eventually points as
it were at some portion of the periphery of the parasite, and very frequently
a condensation of its substance at this point is seen (fig. 1, No. 1). It
is at this period that the blepharoplast is most constantly seen and it very
frequently bears a relation shown in fig. 1, No. 1, an arrangement which
Mr. Fantham tells me he has also observed. As fission approaches, the chro-
matin from the main mass appears to pass over and accumulates in the position
originally occupied by the blepharoplast, thus giving rise to the appearance of two
chromatin masses connected by a bridge of chromatin (fig. 1, No. 2). In
some cases fission would then seem to take place by the two masses separating
and becoming the nuclei of the new parasites. In many cases and possibly in
all a more complicated process takes place*.
After a certain amount of chromatin has accumulated in the new position
it divides into two portions which pass out of the parasite as previously described
(fig. 1, Nos. 4 and 5). At this stage a portion of the original mass is often
still present, the parasite then showing three chromatin masses joined by
delicate threads, the representatives of the extended area (fig. 1, No. 4). Later,
the original chromatin all passes over to one or other of the new masses, some-
times leaving a minute mass which may simulate a blepharoplast (fig. 1, No.
4). A somewhat similar process can also often be detected in the elongated
forms of parasite which divide by a constriction of the centre. In this case the
most common arrangement of the chromatin is for the original mass to be upon
one side about the middle of the parasite, and for the blepharoplast, and later
the new chromatin masses, to be upon the other side, also about the middle
(fig. 1, No. 21). Being so close together the relation of the masses is not so
easy to follow as in the large oval or round forms. In a common method of divi-
sion the blepharoplast arranges itself close alongside the original mass (Pl. 1,
fig. 36). But here the proximity of the chromatin masses usually prevents the
nature of the changes from being followed.
* Since this paper has gone to press Nuttall and Graham Smith have published a very similar account
of these changes. They describe, however, fission of the original mass into two, one of which masses again
divides. The passing over of chromatin and the appearance of the blepharoplast at a certain stage are not
described.
24
In the multiple forms arrangements for a second fission are usually we
advanced very shortly after the preceding division, and no sooner has the
extended area divided into two then, arranging itself transversely to the length of
the parasite, it again "points" and becomes the channel for the separation of
the chromatin (fig. 1, No. 61).
Free forms may show any stage of the above, but most commonly have a
nucleus situated near the pointed end of the parasite with an extended area and
blepharoplast arranged as in the preliminary stages of preparation for division.
In some cases the extended area is apparently at rest and forms a more or less
irregular body lying to one side of the nucleus. In the early invasion forms
division appears to take place after direct division of the large chromatin
mass.
Cultivation forms. —Attempts to observe development of piroplasma in
vitro have been made by several observers. Lignières observed what he consi-
dered a multiplication of the parasite, but others have thought this appearance
to be merely the result of the dissolution of corpuscles leading only to an
apparent increase in parasites. Lignières has also figured two parasites in a cor-
puscle from which projected long and fine flagella, an observation believed by
Doflein to show that flagellation occurs in piroplasma as it does in the malarial
parasite. It is more probable that the flagella figured represented only pro-
cesses of the red cell, such as are frequently seen in fresh films under certain
conditions. Nocard and Motas kept defibrinated blood rich in parasites at 37°C.
They never saw any multiplication, but observed that the parasites became
smaller and rounder, due to a kind of condensation, and that the nucleus retained
its staining properties for a long period, suggesting that the parasites were still
living ; experiments with blood kept fluid in the gut of the leech were equally
unsuccessful in showing any development. These authors note that a very
intense phagocytosis is sometimes seen in vitro.
Nuttall and Smith have made observations on the changes in the blood
from the heart taken immediately after death and kept at room temperature in
the dark. After 24 hours they found the majority of the parasites free in the
serum, whilst others were in the leucocytes. At the end of 48 hours intracorpus-
cular parasites showed very little change in their appearance, but many of them
had very well-marked processes and flagella. Sometimes the parasite occupied
a clear space in the corpuscle. Free forms were numerous and occurred in
groups of 30 or more. The parasites comprising these groups were much
changed being irregular or rounded in shape and varying from minute organisms
one-sixth the diameter of a corpuscle to parasites of a size nearly equal to
that of corpuscle ; but no definite development was observed.
In marked contrast to these results are those of Kleine (187), who following
25
Koch has obtained positive results in vitro with Piroplasma canis. Kleine used
the blood of young pups which had been injected with infective blood. A certain
time after inoculation parasites were found to be very abundant. The dog was
then killed and the blood taken was mixed under sterile precautions with an equal
quantity of normal saline and kept at 27° or room temperature. Under these
conditions the parasites underwent development sending out long processes as
in the development described by Koch as occurring in the tick.
There are possibly several points in favour of cultural results not, so far as
I know, recognised by others who have made cultural experiments.
1. The marked bactericidal effects of the blood noted by Nocard and
Motas is probably minimised by the early date at which the blood is withdrawn,
when puppies are killed so soon as numerous parasites appear.
2. To judge by the susceptibility of very young pups their blood would
appear to be in the most suitable condition for culture.
3. The period of the attack during which the blood is taken has consider-
able importance and in my own experiments disappointing results were some-
times due to the blood having been taken at a time of crisis when an intense
phagocytosis removed practically all the parasites present.
In many of my cultures, though changes occurred in the parasites, it is
doubtful whether any development occurred. Most usually at the end of twenty-
four hours parasites were seen to have become stellate in form (Pl. 1, figs. 45
and 46), sometimes lying in a vacuole in the red cell and sometimes free, but at
the end of forty-eight hours no further changes took place.
In other cultures, especially those taken early in an attack, different condi-
tions prevailed. Forms included in the red cells usually showed at the end of
twenty-four hours either an extraordinary development of flagella-like processes
(Pl. 1, figs. 43 and 44) or remained unaltered. A proportion of the parasites,
especially the free forms, on the other hand exhibited appearances resembling
those of the early changes seen in the tick. They were increased in size and
were often angular or showed projections, but never the long spikes described
by Kleine (Pl. 1, fig. 47).
PROGRESS OF INFECTION.
Infection in the case of Piroplasma canis results from both intravenous and
subcutaneous inoculation of infective blood. The period of incubation as rst
observed by Spreul and Purvis is shorter in the former case. Nocard and
Motas note the onset of fever three to five days after intravenous and five to six
days after subcutaneous inoculation, but parasites have been seen by these
observers so soon as 36 hours after intravenous inoculation, and in a case of
E
26
Nuttall and Graham Smith symptoms appeared only after 10 days. In the case
of subcutaneous inoculation Robertson notes the occurrence of symptoms in
three, four and five days, and Lounsbury six and seven days in two cases.
In my own cases parasites have usually been seen on the 4th or 5th day
after subcutaneous inoculation, and have become numerous the day following
their first appearance, when symptoms are first noticeable. Nocard and Motas
and others have used inoculation as a means of studying the disease, and when
young dogs are used the certainty of infection is as great as that usually
associated with bacterial infections. An interesting point has been raised by
Robertson who, after carrying the disease by inoculation through thirteen dogs,
considered the virulence of the parasite to be enhanced ; my own experiments
are more in favour of an opposite conclusion, and it was noticeable that infec-
tion by ticks gave rise to much more severe types of the disease than did
inoculation. The following series among batches of young dogs of the same
litter inoculated in the course of cultural experiments shows the susceptibility
of dogs to artificial infection and the length of the incubation period under
these conditions.
On 24th August 1906 an emaciated dog showing a fair number of piro-
plasmata was brought to the laboratory. On the same day it was chloroformed
and about half a cubic centimetre of heart blood inoculated subcutaneously into
four young puppies numbered 44, 45, 46 and 47. On the 30th August 1906
dog 44. was seen to be about to die and its blood was found swarming with
piroplasma. At the same time dog 45 had a few large round forms, dogs 46
and 47 being negative. Dog 44 died, and on the 31st August 1906 all the
others showed parasites and were chloroformed on this or subsequent dates.
On the 25th August 1906 a dog showing piroplasmata in the blood, but not
obviously ill, was brought to the laboratory and on the same day four young
puppies numbered 48, 49, 50 and 51 were inoculated subcutaneously with about
half a cubic centimetre of blood. Of these dogs Nos. 50 and 51 were from an
older litter than 48 and 49. On the 31st August 1906 dogs 48 and 49 both
showed abundant large forms and the blood of dog 50 was swarming with
parasites. Dog 51 showed a few forms only, but next day its blood contained
abundant parasites. On the 31st August 1906 two half-grown dogs, Nos. 52
and 53, were inoculated with 1 c.c. of blood from dog No. 45. Dog 52 died on
7th September 1906 and dog 53 was very ill and its bood contained numerous
piroplasmata.
On the same date (August the 31st) two half-grown dogs, Nos. 55 and 56,
were inoculated with.¼ c.c. and 3 c.c. respectively of blood from dog 50. On
the 4th September 1906 dog 55 showed scanty and dog 56 numerous parasites.
The next day both showed numerous parasites.
E 2
TABLE II.—Showing time at which parasites first appeared in the peripheral circulation after the bite of infected ticks.
Number
of dog.
Bitten by
Date bitten.
Piroplasma first seen.
Period of
incubation
in days.
REMARKS.
105
•
Nymphs
. . 13th October 1906 . 17th October 1906 .
4
107
Nymphs
• .
13th October 1906 . 17th October 1906 .
4
109. Nymphs
• .
4th November 1906 . 12th November 1906
8
1 1 0
Nymphs
• .
4th November 1906. 12th November 1906
8
1 1 1
Nymphs
.
•
4th November 1906 . 8th November 1906.
4
112
Nymphs
. . 4th November 1906 . 8th November 1906.
4
121
Nymphs
.
.
13th November 1906 17th November 1906
4
Negative, 16th November 1906.
122
Adults • . .
22nd November 1906 28th November 1906
6
127
Adults . .
29th November 1906 4th December 1906 .
5
129
Adults • . .
29th November 1906 4th December 19 06 .
5
131
Adults .
. 11th December 1906 21st December 1906
1 0
First infection probably earlier as not examined previously,
132
Adults.
• .
11th December 1906 21st December 1906
1 0
Ditto
ditto.
135
Adults .
.
. 10th December 1906 18th December 1906
8
Swarming piroplasmata when first examined December 1906.
but free on 15th.
136
Adults .
. . 10th December 1906 18th December 1906
8
Ditto
ditto.
139
Adults . .
. 10th December 1906 18th December 1906
8
Ditto
ditto.
140
Adults .
.
.
10th December 1906 18th December 1906
8
Ditto
ditto.
28
On the afternoon of 22nd October 1906 half-grown dogs Nos. 98 and 99
were inoculated in each case with ½ c.c. of infective blood. On the morning of
the 26th October 1906 scanty piroplasmata were found in dog 98, but none in dog
99. Next morning both dogs showed infection.
Under conditions of natural infection by ticks the incubation period has by
most observers been placed at from 10 to 15 days. Lounsbury in his long series
of feeding experiments with infected H. leachi has usually observed incubation
periods of from 10 to 12 days, but in some cases the period was extended to
14 and even 21 days. In Nuttall and Graham Smith's cases the incubation
periods in the cases of three dogs infected by ticks were 13, 15 and 16 days
respectively.
The periods differ very markedly from those in my own experiments in
which infection was brought about by means of numerous recently and heavily
infected R. sanguineus. As will be seen from the table the time for the
appearance of parasites in some cases has been so short as four days. In one
or two instances a much longer incubation period was noted, but these cases
occurred before rigid precautions had been taken to exclude chance infection
and cannot be relied upon as can those which occurred in the course of
experiments given in full later on, and in which the possibility of error was
eliminated by precautions to be described. That dog after dog succumbed to
infection at or about the same time after exposure to the bite of infected ticks
whilst controls remained well throughout can scarcely be interpreted in any
other way than that, under the conditions then present, the naturally produced
disease followed extremely quickly upon the bite of the infecting ticks. The
following list of infections acquired naturally in the laboratory also points to an
extremely short incubation period in the disease under certain circumstances.
The cases occurred among young healthy pups brought to the laboratory and
allowed to run about at a time when the laboratory was infested with ticks derived
from dogs used in earlier investigations. It was of course possible that the dogs
had already contracted infection from outside, but as they came from various
sources and all sickened about the same time after admission this is not likely.
Infected dogs were first admitted to the laboratory about the beginning of
October 1905, and shortly afterwards the walls were observed to be dotted over
with gorged larvæ, nymphs, and adults. On the 22nd October 1905 newly
hatched nymphs were seen and a little later adults.
Dog 5. Young pup—admitted 22nd, October 1905.
26th October 1905. Many gorged nymphs evidently derived from the labor-
atory seen on dog.
27th October 1905. Dog very sick. Complete anorexia and prostration.
Piroplasmata swarming.
28th October 1905. Dog died in the night with hæmoglobinuria.
29
Dog 6. Pup—admitted 22nd October 1905. No piroplasma.
28th October 1905.
Scanty piroplasma.
29th October 1905. Many large round forms.
5th November 1905. Moderate infection with pear-shaped forms.
Developed a mild infection.
Dog 7. Pup—admitted 23rd October 1905. Moderate if any ticks. In good condi-
tion. No piroplasma.
28th October 1905. Dog in good health.
30th October 1905. Quiet and off colour. A moderate number of large
round forms seen.
31st October 1905. Many parasites.
1st November 1905. Large forms plentiful. Dog quiet but not very ill.
Dog 13. Pup—admitted 12th November 1905. Blood examination negative.
18th November 1905. Scanty piroplasma.
Dog 14. Pup in good condition and free from ticks. No piroplasma. Admitted
1 8 t h N o v e m b e r 1 9 0 5 .
29th November 1905. Very ill; piroplasma abundant.
Dog 17. Pup in good condition. Admitted 21st November 1905. No piroplasma.
30th November 1905. Slightly indisposed. Piroplasma scanty.
Dog 18. Pup—admitted 19th November 1905. Extremely numerous nymphs and larvæ.
No piroplasma.
26th November 1905. Abundant piroplasmata.
Probably infected from nymphs picked up a few days
before admission.
Dogs 24, 25, 26, 27 and others kept protected from ticks preparatory to feeding experi-
ments did not develop infection, nor in later experi-
ments did healthy young pups of this class develop
infection when kept under protective conditions in the
laboratory.
The general progress of the disease has been indicated; but the exact
relationship of the parasite to the disease requires further discussion. Nocard
and Motas note febrile periods characterised by the presence of numerous
parasites which exhibit sign of amœboid activity and multiplication, and a
subsequent period in which parasites are scanty and do not show amœboid
movements.
They also speak of a hæmoglobinuric crisis. Other authors have paid little
attention to periodicity in the disease, it being generally taken for granted that
development of the parasite is irregular and devoid of periodicity.
During the examination of different cases it was often observed that imme-
diately following an outburst of parasites there was a time during which parasites
almost disappeared from the blood and that there followed, usually at an interval
of about twenty-four hours, another outburst of parasites; and later observations
have shown that there is in canine piroplasmosis a condition closely resembling
30
the periodicity of malaria in man. If examinations be made every few
hours there will be found as a rule at the same time of the day, usually in the
morning, a period during which parasites are very numerous, red cells infected
with four, eight or sixteen forms and groups of free forms being conspicuous.
F o l l o w i n g s u c h a p e r i o d t h e r e o c c u r s i n t h e g r e a t m a j o r i t y o f c a s e s a t i m e
when parasites are few or even in some cases apparently absent from the
peripheral circulation and when multiple forms are difficult to find. This
gives place again to an increased number of parasites and the occurrence of
another attack. As a rule the interval between two such attacks is, as previously
stated, about 24 hours, but it is not infrequent to find what appears to be a double
infection present, one attack taking place in the morning and another some time
in the evening. In severe cases the periods of attack may run into one another
giving rise to more or less continuous infection; but especially towards the
beginning and end of the initial outbreak the cycles are short and separated by
well-marked intervals during which parasites are scanty. In the chronic disease
short attacks lasting a few hours occur, parasites in the intervals being extremely
scanty.
It was thought at first that the periods represented a cyclical development of
the parasite and that the formation of eight and sixteen forms was analogous to
sporulation of the malarial parasite. But the result of counts of parasites already
given showed that this view required considerable modification, especially when
it was observed that at the critical period, when free forms became a conspicuous
feature, these were derived not only from corpuscles containing large numbers of
parasites, but also from corpuscles containing two and four forms.
The nature of the process seemed therefore to demand careful observation,
and a number of cases were followed very closely throughout several days to
determine what changes actually took place. That the number of parasites
undergoes a great increase at the time of attack has already been noted, and the
charts at the end of this memoir demonstrate its extent. It was also found that,
though the relative frequency of the different forms remained in the main un-
altered, there was at the time of crisis a distinct increase in the percentage of
corpuscles containing four and more forms; this also will be apparent from the
charts. In the case of the single and double forms distinct alterations in their
proportions also took place, there being times when the single forms preponder
- a t e d v e r y g r e a t l y o v e r t h e d o u b l e f o r m s , a n d o t h e r t i m e s w h e n t h e l a t t e r e q u a l l e d
or even excelled the former in frequency. Unfortunately in the case of the single
and double forms it has not been possible to demonstrate accurately the relation
of the changes to the attack. On the whole a distinct impression was obtained
that a large increase in the relative frequency of single forms together with an
absolute increase of total parasites was usually the first sign of a coming attack,