NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
ON A PARASITE FOUND IN THE WHITE CORPUSCLES OF THE
BLOOD OF DOGS.
SO far as I am aware there is no record in literature of the discovery of para-
sites of the white blood corpuscles of mammals. For this reason I am
led to record some observations I have made upon a leucocytic parasite which is
not uncommon in the peripheral blood of dogs in Assam.
Although I do not consider that the parasite I am about to describe has any
connection with the leucocytic parasites of birds described by Danilewsky,
Sacharoff, Ziemann, and others, it will not be out of place if I refer briefly to the
work of those observers, for parasites of the leucocytes have been discovered
so rarely that our knowledge regarding them is very slight.
Danilewsky 1 discovered a parasite in the white blood corpuscles of a
screech-owl between 1884 and 1886. He studied it only in fresh preparations.
In his first communication he described it as a spherical or oval lightly granular
body contained in a delicate transparent homogeneous envelope provided with a
large biscuit-shaped nucleus. He did not describe a nucleus as being present in
the parasite itself and evidently was interested chiefly in the nature of the mass
in which the parasite was contained. From the size and shape of this envelope,
the absence of grains of melanin in it, and the appearance and size of its nu-
cleus he concluded that it was a white blood corpuscle—that the parasites
were, in fact, Leucocytozoa. He observed frequently that the body-substance of
the leucocytes in which the parasites were contained was drawn out in the form
of triangular tags and for this reason concluded that the leucocytes were in a state
of degeneration. Each parasite was described as being more than 1½ times
as large as a red blood corpuscle.
In his second communication, published in 1890, he described the cells
containing parasites as having the appearance of large fusiform bodies with a
central granular portion and an elongated excentrically situated nucleus. The
granular mass was sometimes spherical in shape. He repeated his opinion
that the parasite was contained, not in a red blood corpuscle, but in a degen-
erated leucocyte, and stated that he had been able to show that it was only
an intracellular stage in the development of the flagella bodies ( Polimitus)
previously described by him as being present in the blood of birds. He consi-
dered, however, that the flagella bodies produced from the leucocytozoon differed
from those which develop in red blood corpuscles in being of larger size and in
having no granules of melanin in their interior. In the bone-marrow he observed
young stages of the parasite as small oval or spherical bodies enveloped by an
2
almost homogeneous layer of leucocytic protoplasm which had lost the power of
amœboid movement. The nucleus of this envelope was large and distinct and
almost always much elongated; if the parasite was of considerable size it pressed
upon the nucleus and caused it to become biscuit-shaped. In attempting to
explain why the parasites are not destroyed by the phagocytic action of the cells
in which they are contained he threw out the suggestion that possibly these cells
are not, after all, fully developed leucocytes but erythroblasts.*
In a third paper he described in more detail the development of flagella
bodies from a leucocytozoon found in the blood of a grey crow. In this case the
parasite was described as a large round finely granular body containing a small
clear nucleus.
Sacharoff 2 described parasites of the leucocytes as being present in the
blood of crows, rooks, and magpies. In crows they were present along with
parasites of the red blood corpuscles. He examined them both in fresh
preparations and in preparations stained by Romanowsky's method. In fresh
preparations a nucleus could not be seen in the parasites, but, after staining,
a faint rose-coloured nucleus with an indistinct contour could be made out.
He described two forms as being present in the blood of crows, viz., (1)
forms containing numerous deeply staining granulations and some vacuoles;
and (2) homogeneous forms. Flagella bodies were frequently observed to
originate from the homogeneous parasites, but very rarely from the granular ones.
He considered that the parasites of the blood of crows pressed upon and gra-
dually destroyed the nuclei of the leucocytes in which they were contained and
for this reason regarded them as a form of Karyophagus. He described the
youngest forms as being found in the blood of rooks. They were oval or
fusiform bodies of the size of an eosinophil granule, and were usually present in
lymphocytes, but sometimes free in the plasma. They possessed a small
nucleus. He considered that the parasites found in rooks differed from those
found in crows in that they did not destroy the nuclei of the cells in which they
were contained.
In 1898 Ziemann 3 discovered similar leucocytic parasites in the blood of
three owls. He examined them chiefly in preparations stained by a modifica-
tion of Romanowsky's stain. He described three forms, viz., (1) round
or oval sharply outlined parasites from 2/3 to the full size of a red blood cor-
puscle. These were free in the plasma. In stained preparations they presented
a compact round or elongated nucleus, and in their body protoplasm a stained
outer zone and an unstained, or less deeply stained, inner zone could nearly
* The word used by Danilewsky is hématoblaste; it must not be confounded with the English word
hæmatoblast. For definition of the latter term and of erythroblast see Quain's Anatomy. 10th edition,
vol. I, part II, pages 215 and 219.
3
always be made out ; (2) forms resembling these but enclosed in a sharply out-
lined mass (leucocyte) possessing numerous small granulations. The body
of this form stained deeply and contained dark-staining granules and light un-
stained spots. Its nucleus was round and stained deeply. The leucocyte in
which the parasite was contained was often shaped like a whetstone and oc-
casionally reached a length of 44 micromillimetres: its nucleus was dumb-bell-
shaped and partially encircled the parasite; (3) round forms two or three times
as large as the previous form with larger and more numerous clear spots.
Ziemann did not observe multiplication stages of any kind and in the internal
organs of two owls upon which post-mortem examinations were made the para-
sites presented the same appearances as in the peripheral blood. He regarded
the parasites found by him as different from those found by Danilewsky.
Berestneff 4 studied leucocytic parasites in an owl, a crow, and a magpie. In
the owl and crow they were present together with Halteridium danilewskyi and
in the crow filariæ also were present. In the magpie no other parasites were
seen. In fresh preparations of the owl's blood the leucocytozoon was present in
long spindle-shaped and round forms. The spindle-shaped forms contained
numerous small round granules and a clear round nucleus ; the round forms had
the appearance of a large ring containing dull protoplasm in which an oval nu-
cleus was seen. Almost the whole of the latter form of the parasite was encircled
by the nucleus of the leucocyte. In preparations stained by Romanowsky's
method the body of the spindle-shaped forms stained intensely blue and contained
small deep violet-coloured granules and unstained round spots. The nucleus
was usually oval in shape and only faintly stained : near it lay a deeply coloured
round or irregularly shaped "nucleolus" of about .7 to 1.5 micromillimetres in
diameter. The second form was about the length of the diameter of a red
blood corpuscle. Its body-substance stained faintly and was homogeneous
or only slightly granular. The nucleus was considerably larger than that of
the first form. The nucleus of the leucocyte was closely applied to the parasite
and often was of an irregularly quadrilateral shape.
Young stages of the second form of parasite were seen by Berestneff in
mononuclear leucocytes (large lymphocytes). Their protoplasm was faintly
stained and contained a large nucleus and some deep blue granules. He regarded
both types of the parasite seen by him as sexual forms, those with feebly
staining body-substance and a large nucleus being the males, and those with
deeply staining body-substance, a small nucleus, and a "nucleolus," being the
females. He was unable to confirm the observations of previous workers regard-
ing the development of flagella bodies from the parasites.
Laveran5 and Schaudinn 6 have also studied these leucocytozoa in the blood
of owls. Laveran described a female form and an immature male form observed
4
in the blood of an owl, Syrnium aluco . Neither form contained pigment. In
opposition to Ziemann and other observers he regarded the elongated, spindle
-shaped elements in which the parasites were included, not as leucocytes, but as
deformed red blood corpuscles with hypertrophied nuclei. He considered that
the male and female gametes carried out fertilization in the same manner as mem-
bers of the genera Plasmodium and Halteridium and therefore regarded the
parasites as Hæmamœbæ, naming them Hæmamœba ziemanni .
Before referring to Schaudinn's work upon these parasites it will be well to
summarize in the following brief manner the somewhat contradictory results out
-lined above. In addition to the well-known pigmented parasites Halteridium and
Proteosoma , there have been found in the blood of certain birds unpigmented
nucleated parasites which may be present in two forms, viz. , (1) a deeply staining
granular (female) form containing a "nucleolus" as well as a nucleus ; and (2) a
feebly staining homogeneous (male) form. In the peripheral blood of the birds
both these forms usually have been observed to be contained in, or attached to,
elongated nucleated elements which Danilewsky, Sacharoff, Ziemann, and Berest-
neff regarded as leucocytes, but which Laveran thought were deformed red blood
corpuscles. Danilewsky, however, in one of his papers, threw out the sugges-
tion that some of the elements in which the parasites were contained were
possibly erythroblasts, and Sacharoff, as regards the cells in which the parasites
of the blood of rooks (but not of crows) were contained, thought this suggestion
very probably correct. Several of the observers described the production of
flagella (microgametes) from one or other form of the parasite when the blood
was examined in fresh preparations. Danilewsky termed the parasites Leucocyto-
zoa ; Sacharoff and Berestneff looked upon them as being probably a form of
Karyophagus ; and Laveran considered that they were true Hæmamœbæ .
At this stage of knowledge Schaudinn commenced his study of these
parasites. He observed the two forms seen by previous investigators in the
peripheral blood of owls and confirmed the correctness of Laveran's opinion
that they are male and female sexual forms which, after their exit from the body
of the bird, undergo development in the same manner as parasites of the
genus Plasmodium, i.e ., by the production from the male form of flagella
(microgametes) which fertilize the female form or macrogamete, the resulting
body being an ookinet. He recorded the following additional facts as a result of
his researches. The formation of the microgametes and the fertilization by them
of the macrogametes normally takes place in the mid-gut of a "culex" mosquito.
The ookinet which results becomes greatly elongated and its nucleus divides up
into a large number of smaller nuclei each of which becomes surrounded by a
dense zone of plasma. From the cellular areas thus formed there are developed
a number of trypanosome-like flagellates which separate themselves from the
5
residual body, become much elongated, and develop into typical spirilla. These
spirilla migrate into the malpighian tubes of the mosquito and undergo multipli-
cation by longitudinal division. They are to be considered as having periods of
rest and periods of movement. During the former they enter the cells of the
malpighian tubes ; during the latter they become free, multiply by longitudinal
division, and after having flooded the malpighian tubes reach the anterior part of
the œsophagus of the mosquito whence they are again transferred by means of
the bite of the mosquito into the blood of the first host. Here they undergo
asexual multiplication in the form of spirilla and are ultimately transformed into
the sexual stages (the so-called leucocytozoa) from which their cycle of develop-
ment commenced. In the body of the bird as in that of the mosquito they are
to be regarded as having periods of rest and periods of movement. In the blood
of the spleen and bone-marrow such of the flagellates (spirilla) as are to become
sexual forms undergo a remarkable increase in size and attach themselves by
their posterior extremities to certain elements which Schaudinn considers to be
erythroblasts. They then pass into a period of rest and gradually absorb into
their substance the body of the erythroblast. At this stage they may appear in
the peripheral blood, and it is in this condition that they were discovered by
Danilewsky and others. After remaining in the stage of rest for a certain time
the flagellate apparatus is formed anew, the nucleus of the erythroblast is cast
off, and the parasite enters again upon a period of movement in the form of a
trypanosome-like flagellate. The periods of rest and of movement alternate.
Growth takes place during the former, multiplication during the latter.
The so-called leucocytozoa discovered by Danilewsky and studied by
Sacharoff, Ziemann, and others are, therefore, according to Schaudinn, the rest-
ing sexual stages of his trypanosome-like parasite, the nucleated mass in which
those observers thought the parasite was contained being, in his opinion,
neither a leucocyte nor a red blood corpuscle but an erythroblast to which the
parasite has attached itself and which (with the exception of the nucleus) it
absorbs into its own substance. The " nucleolus" observed by Sacharoff, Berest-
neff, and Laveran in one of the forms is considered by Schaudinn to be a true
blepharoplast.
The cycle of development undergone by these so-called leucocytozoa is
therefore, according to Schaudinn, very similar to that of Halteridium except
that, while the resting stage of the latter parasite is passed at the expense of the
red blood corpuscles that of the former is passed at the expense of those forms
of white cells known as erythroblasts. For this reason their development, while
in the bodies of the birds, is carried on chiefly in the bone-marrow. In other
ways the details of development of the leucocytozoa are somewhat different from
that of the halteridia, and Schaudinn regards them as belonging, not to the genus
6
Trypanosoma, but to the genus Spirillum, and names them Spirillum
ziemanni.
Z i e ma n n 7 n o w a g r e e s wi t h S c h a u d i n n i n r e g a r d i n g t h e s o - c a l l e d l e u c o c y t o z o a
found in the peripheral blood of owls as stages in the development of a trypano-
some-like organism and Edmond and Etienne Sergent 8 h a v e p a r t i a l l y c o n f i r m e d
Schaudinn's observations.
So far as I can ascertain, the above is a brief summary of all that is known
regarding parasites of leucocytic blood cells, and now I shall describe the parasite
I have studied in the blood of dogs. Observations upon this organism are simpli-
fied by the fact that there can exist no doubt regarding the nature of the
anatomical element of the blood in which it is contained.
In June 1904 Dr. C. A. Bentley, of Borjulie, Assam, informed me that he had
found a peculiar body in the leucocytes of the blood of a dog. He thought
the body might be a parasite of some kind, but, with the stain he was then using
he had been unable to bring out any details of structure in it. He showed me
a slide in which some of the leucocytes contained a body having the appearance
of a large regularl y shaped vacuole. In a few of the bodies a very faintly stained
nucleus could be seen. Fortunately Dr. Bentley knew the owner of the dog
in the blood of which the bodies had been found, and the next day we were able
to procure more slides from it and to study the parasite together for some days.
Afterwards I made a practice of examining the blood of such dogs as I was able
to obtain in the places I visited, with the following results:—
p l a c e .
Number of dogs
examined.
Number in which
the parasite of
the leucocytes,
was found.
Remarks on the presence or absence of other
parasites.
Borjulie .
.
.
.
1 6
2
One dog in which the leucocytic parasites
were not found had filarial embryos in its
bl ood.
Gauhati .
.
.
.
3
1
Piroplasma
canis was also present at one
period of this case.
Tezpur
.
.
.
.
5
2
In one of the two cases filarial embryos
were also present.
Silghat .
.
.
.
7
0
......
Nowgong
.
.
.
8
1
No other parasites present.
Dacca (Bengal)
.
.
6
0
. . . . . .
I have therefore found the parasite in the peripheral blood of six out of
4 5 d o g s e x a m i n e d ; i n f o u r c a s e s i t w a s p r e s e n t a l o n e , i n o n e c a s e i t w a s p r e s e n t
together with filarial embryos, and in one case with parasites of the red blood
corpuscles (Piroplasma canis).
The number of parasites present in films of the peripheral blood varied
7
greatly in the different cases. The following results of differential counts of the
leucocytes made in four cases will show this, as well as the relative proportions of
the different varieties of leucocytes present :—
Case 1.—Number of leucocytes enumerated = 600.
Number of parasites seen = 35.
Relative proportions of the different varieties of leucocytes:—
Polymorphonuclear cells
67 per cent.
Large mononuclear cells
3 per cent.
Small mononuclear cells
29 per cent.
Eosinophil cells
1 per cent.
Case 2. —Number of leucocytes enumerated = 1,165.
Number of parasites seen = 58.
Relative proportions of the different cells :—
Polymorphonuclear cells
58.2 per cent.
Large mononuclear cells
4.1 per cent.
Small mononuclear cells
36.4 per cent.
Eosinophil cells
1.1 per cent.
Case 3. —Number of leucocytes enumerated = 500.
Number of parasites seen = 16.
Relative proportions of the different cells:—
Polymorphonuclear cells
65 per cent.
Large mononuclear cells
3.4 per cent.
Small mononuclear cells
30.2 per cent.
Eosinophil cells
1.4 per cent.
Case 4. —In this case filarial embryos were also present.
Number of leucocytes enumerated = 590.
Number of parasites seen = 6.
Relative proportions of the different cells :—
Polymorphonuclear cells
65.7 per cent.
Large mononuclear cells
2.8 per cent.
Small mononuclear cells
22.4 per cent.
Eosinophil cells
8.9 per cent.
In the first and second cases one parasite was seen to about every 11 poly-
morphonuclear cells, in the third case one parasite to about every 20 polymor-
phonuclear cells, and in the fourth case one parasite to about every 64 polymor-
phonuclear cells.
A special feature of all the films was the presence of nucleated red blood
corpuscles in considerable numbers.
I have had but few opportunities of examining the parasites in fresh prepara-
tions, and I shall therefore confine myself chiefly to a description of them as they
appear in films fixed in alcohol and stained by Romanowsky's method.
In the first place it should be mentioned that in films quickly made in the
ordinary way from a drop of blood removed from the foot or ear the parasites are
almost invariably contained in polymorphonuclear leucocytes. During the
8
examination of 2,855 leucocytes in films from the four cases mentioned above as
well as during less minute examinations of slides from two other cases, I have
seen only one parasite which was free in the plasma ; every other was contained
in a polymorphonuclear cell.
In the second place it is necessary to mention that in order to bring out the
structure of the parasites distinctly the films must be well and deeply stained.
When this has been done the parasites are seen very clearly and cannot easily
be passed over during the routine examination of a slide. As will be seen from
the drawings at the end of this paper the chief characteristic of the bodies as
they present themselves during the examination of a stained film is their
remarkable uniformity in size and appearance. They are usually about 1½ times as
long as the diameter of a red blood corpuscle and the majority are almost ex-
actly twice as long as they are broad. The shape of the body may perhaps be
best compared to that of a bean.
On careful examination of a well-stained film it will be seen that these
characteristic bean-shaped bodies present in the leucocytes consist of two parts,
viz., (1) a definite oblong almost quadrilateral capsule or cytocyst, the outline of
which is often coloured bright pink with Romanowsky's stain ; and (2) a parasite
enclosed within this capsule.
The capsule or cytocyst would appear to be formed from the protoplasm of
the leucocyte as the result of the presence of the parasite. It is thick and evidently
resists considerably the penetration of the stain through it, for unless prolonged
staining has been employed, the parasite itself remains uncoloured and the
appearance presented is like that of the body shown in one of the leucocytes in
figure 7. The sides of the capsule are often pressed inwards and folded over so
that in stained films they appear as in figure 10.
The parasite itself is inside the capsule. This it often completely fills
and in such cases its outline cannot be clearly differentiated from the outline
of that structure (Figs. 1, 2, 3, 4, etc.). In many cases, however, it is somewhat
retracted or shrunken so that there is a clear space between its circumference
and that of the capsule (Fig. 5). In these cases its shape and characteristics
are clearly seen. It is an oblong bean-shaped body composed of a homogeneous
or very slightly granular protoplasmic body provided with a large oval or round
chromatin nucleus. Its body-substance stains a faint sparrow-egg blue colour
and its nucleus, which is usually situated near one end, stains in the same
manner as the nuclei of the leucocytes. The parasite has a clearly defined
circumference but apparently is not surrounded by a definite membrane, and
its appearance is such as to indicate that when not imprisoned within its
leucocytic capsule, it has the power of movement. It contains no grains of
pigment.