NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
90
Streptotrichosis.
Photograph No. IV.—In this case the parotid glands were
the seat of abscesses. The bullock recovered.
CULTURES.
The first cultures from fresh material were difficult to ob-
tain, and growths of the organism appeared in less than five
per cent. of the inoculated tubes, and only after three or more
weeks. Pure growths were obtained on Agar and potato from
pus from the affected glands. In subcultures, growth took
place more rapidly. Also passage through experimentally
inoculated rabbits and guinea-pigs increased the rapidity and
abundance of growths in culture media to such an extent that
cultures were visible 48 hours after inoculation. In the
experimentally inoculated small animals, pure cultures were
recovered both from the pus and the heart blood.
From the affected glands received from the Calcutta
bullocks, several pyogenic organisms as Staphylococci aureus
and albus and Bacillus pyocyaneus were frequently isolated
in culture. The cultures of the Streptothrix presented great
variations. Some cultures obtained from the heart blood of a
rabbit, dead a week after inoculation from a culture, when
first grown on potato, after 48 hours, gave a peculiar reaction.
The potato had a macerated appearance and showed on the
surface a distinct reddish colour. Subcultures from this grew
in a more characteristic manner as dry, brown-coloured
pellicles. Other cultures on potato produced white, brown
or yellow colonies without any alterations of the medium.
At times the growths in Agar were of pure white colour,
either in the form of single colonies of button shape, with cre-
nated edges and a raised centre of brown colour, or as a uniform
surface growth, with a downy appearance, much resembling
a mould. The growth sunk into the medium, and was tough
and adherent.
Growth of the organism takes place at 20°C. and at 37°C.
and both aerobically and anaerobically.
Aerobic conditions at a temperature of 37°C. are the more
favourable.
Agar surface.—The organism grows best on glucose or
glycerine Agar. The early growth resembles dew drops on
PHOTOGRAPH No. I.—See page 89.
PHOTOGRAPH No. II.
A Bad Case. All over the animal contained abscesses. —See page 89.
PHOTOGRAPH No. III.—See
page 89.
PHOTOGRAPH No. IV.—See page 90.
Streptotrichosis.
91
the surface of the Agar. Following this, a thin dry skin forms
of a dirty white colour. This pellicle gradually increases in
depth, and becomes corrugated and of a brown colour. In
the older growth masses of dry, brownish yellow material be-
come heaped up on the surface of the ridges. Single colonies
under a low magnification show a fringe of thread-like projec-
tions round their edges.
Agar stab.—Small colonies develop along the line of
inoculation, but their growth is limited. At the surface, growth
is more abundant and forms a heaped-up centre. At the point
of stab, a wrinkled pellicle forms over the whole surface.
Gelatine.—The growth is much the same as on Agar, but
is less luxuriant. The medium is not liquefied. In old cul-
tures, liquefaction was observed.
Bouillon.—The organism grows well, in plain glucose,
lactose, glycerine, nas and phenyle broth. The growth on
the third or fourth day appears on the surface as floating parti-
cles which, later, form into a thick, corrugated pellicle, greyish
white, adherent to the sides of the tube and growing down for
1/8 to ¼ inch into the medium. The under-surface has a honey-
comb appearance. The broth remains clear. After long
incubation ball-like masses form at the bottom of the tube.
In Nas broth a pinkish tinge was observed in the lower
part of the surface growth.
In neutral red broth, the colour is unchanged.
Milk does not coagulate, but is slowly peptonised.
In litmus milk, there is an acid reaction.
Potato.—As a rule, the growth is similar to that on Agar
surface, but a wrinkled brown pellicle with heaped-up masses,
whilst at times the colour varies to pure white, or yellow.
Staining.—The streptothrix stains with all the ordinary
dyes. It is stained by the Gram and Gram Weigert method,
but in preparations from fresh material and also from cultures
the filamentous form loses the stain if the decolorising process
is prolonged.
It is not acid fast.
Microscopic appearance.—The organism presented itself
under various forms. In some preparations it showed as a
mass of branchy filaments. The dimension of these filaments
92
Streptotrichosis.
was very variable. From potato and agar cultures they were
much thicker than from growths in broth. Numerous rods
were present with these filaments. Many of these rods did not
take the stain well and in their centre one or more deep-stained,
small, spore-like bodies were visible. These were of the same
size and appearance as the mass of cocci-like bodies seen in
preparations from the fresh material. In some old cultures
filaments with club termination were found.
In other preparations, no filaments were present and they
consisted of rods and a large ovoid cell taking the stain at the
periphery and at either end.
ANIMAL EXPERIMENTS.
Twenty guinea-pigs and the same number of rabbits were
inoculated with an emulsion of pus from the affected glands.
The pus was taken from three cases, and separate animals were
inoculated from each. The pus was broken up in normal salt
solution, and the amount used for injection varied from 0.5 c.c.
to 1 c.c. Injections were made subcutaneously and intraperi-
toneally. In the animals which received subcutaneous
inoculations a nodule from the size of a pea to a marble appeared
at the seat of injection after two to four weeks. The affection
was not progressive and the abscess showed no signs of softening
or of spontaneous evacuation. The nodule contained pus
with the same macroscopic and microscopic characteristics
as the original material, and from it pure cultures of the
organism were recovered. From cultures obtained from the
lesions in guinea-pig and rabbits inoculated from the fresh
material, some 20 more rabbits and guinea-pigs were inocu-
lated. These succumbed rapidly in from two to seven days
without showing any outward symptoms. From the heart
blood cultures similar to the original were recovered.
The small animals inoculated intraperitoneally with emul-
sions from the fresh pus from bullocks died after from two
to six weeks. In the majority of these cases, no visible
lesions could be found, but from the heart blood pure cultures
were in several instances obtained. In two guinea-pigs and
three rabbits, miliary abscesses were found post mortem.
Some of these abscesses were adherent to the peritoneum and
PLATE B.
Streptotrichosis.
93
others scattered throughout the mesentery. The pus contain-
ed was of the same color and appearance as the original and
contained similar characteristic masses, and from it pure cul-
tures were grown, which, when inoculated into rabbits and
guinea-pigs, killed in a few days; and from the heart blood
of some of these animals typical pure cultures were recovered.
Inoculations of pus emulsion into two bulls subcutaneously
and two intraperitoneally gave negative results.
DESCRIPTION OF PLATES A AND B.
PLATE A.
Drawn with 2.0 mm. Apert. 1.30 Zeiss. Oc. 4. Stained by Gram.
The material from which (a, h, e, f) were taken was forwarded by
Captain Edwards from the cases among Transport Bullocks, (b & c) from
rabbits inoculated with fresh material, (d & f) from rabbits inoculated from
a pure culture isolated from fresh material and also recovered from the
inoculated rabbits—
(a)
Cocci and peripheral clubs from pus (bullock).
(h)
Cocci and filaments from pus (bullock).
(e & f)
Clubs ray formation from scrapings from a capsule of an
abscess (bullock).
(b & c) Cocci and filaments from pus (rabbit).
(d & f)
Cocci and filaments in striated arrangements from pus
(rabbit).
PLATE B.
Drawn with 2.0 mm. Apert. 1.30 Zeiss. Oc. 4.
No. I.—From a pure culture, grown in plain broth taken from the pus
of an affected lymphatic gland (bullock), shows rod and
thread forms.
No. II.—From a culture grown on Nas agar, isolated from an abscess in a
bronchial gland (bullock), shows rods, spores and filaments.
No. III.—Cultures from a rabbit, inoculated from culture I. Rods, spores
and filaments. Threads show true branching, and also beaded
appearance.
No. IV.—Taken from a sub-culture of No. III on Agar. This culture
presented a white downy appearance.
PART VI.
FLAGELLATE FORM OF PIROPLASMA BOVIS.
Several authors have recorded flagellate bodies observed
in the blood of animals suffering from Piroplasmosis.
Bowhill and Le Doux (1) give a description of flagellate
forms in the blood of a dog which had died of Piroplasmosis
canis. The blood films were made at the post-mortem exam-
ination. Several endoglobular parasites were observed which
showed long flagella-like processes, some with two bulbs on the
flagellum and some with only one at the end.
Their illustrations also show free flagella-like bodies with
a bulb at both or one end. In some instances, the authors
thought they observed flagellated bodies entering infected
corpuscles.
In the same article mention is made of similar forms seen
by Bowhill in the blood of cattle affected with Rhodesian Fever.
Lignières (2) had previous to this described flagellate forms
in the blood of cattle suffering from La Tristeza. He noted
globules with flagella which he considers simple prolongations
of the protoplasm of the cell. One observes them in blood
recently taken. They form on a heated slide. Some of these
flagella were seen free with a thickening at one end. They
separate from the globule and traverse the field of the miscros-
cope. He suggests that these flagella are the same as those
described by Krogius, Von Hellens, San Felice and Lodovico,
as prolongations of the parasite and analogous to the flagella
of the spherical bodies of the hæmatozoa of Laveran. Lignières
does not accept this view, as he has seen the flagella develop
from the red cells and also because they stain with eosin.
In equine Piroplasmosis, Bowhill (3) has seen free pear-
shaped bodies with a long flagellum ending in a bulbous pro-
tuberance. Flagellate forms were also observed where the
body and flagellum were situated within a blood corpuscle.
Piroplasma Bovis.
95
Nuttall and Graham Smith (4) in their study of Canine
Piroplasmosis met with flagellate forms in blood. Free para-
sites with short processes protruding from the pointed extrem-
ity were occasionally seen, but forms with long terminal
flagella were seldom recorded. They apparently did not ob-
serve free flagella as described by Lignières, Bowhill and others.
Schaudinn (5) who studied specimens prepared by Weber
from a cow, two hours before death, from Piroplasmosis,
found typical examples of the Piroplasma Bovis and also
trypanosome-like forms of smaller size than T. Evansi. He
considered these forms related to the Piroplasma.
Schaudinn, therefore, concluded that the P. Canis and P.
Bovis multiply in the same way as the Hæmoproteus Noctuæ
possessing trypanosome and spirochæte-like stages.
Kossel and Weber found similar trypanosome-like bodies
in the gut contents of ticks taken from cattle suffering from
Piroplasmosis.
Koch, Kleine, Kinoshita and Miyajima frequently observ-
ed flagellate shapes of Piroplasma in cultivation forms and in
developmental forms in the tick.
In a recent publication, Breinl and Hindle (6) described fla-
gellate forms of the P. Canis. These were repeatedly observed
in the peripheral blood of dogs on the day before death. These
authors are of opinion that these forms of P. Canis represent
a very transient stage in its life-history. In the blood of cattle
in India, I have on several occasions been able to observe
flagellate forms in connection with Piroplasma Bovis. In
this Laboratory where a very large amount of Rinderpest Anti-
Serum is prepared, a small percentage of the serum-preparing
cattle succumb during the process of hyper-immunizing to a
complication of Trypanosomiasis or Piroplasmosis and fre-
quently to a combination of both diseases. The massive injec-
tions of Rinderpest blood have the effect of lowering the resis-
tance of the system to these two diseases which exist in a latent
form in the majority of cattle of this country. In such cases
the animal may die suddenly without having shown any
clinical symptom of illness or may be off feed, wasting and lying
down for several days before death. It is often necessary to
96
Piroplasma Bovis.
examine the blood repeatedly before finding the Trypanosomes
or Piroplasma.
In the blood of cattle suffering from Piroplasmosis one or
two days before death or immediately after death, numerous
free flagella are frequently to be found.
These forms (Plate C a) are difficult to distinguish in the
fresh blood, but can easily be studied in stained films. They
very much resemble the free forms described by Bowhill and
Lignières. They vary in length from less than half the
diameter of a blood corpuscle to 3 times this length. At one
or both ends, is a slight protuberance in which a cromatin
staining body can often be detected. Numbers of these
flagella can be seen free, moving in the field, others appear to
be entering blood corpuscles, while others seem to be leaving
or to be attached to degenerated and breaking down red cells
A mononuclear leucocyte is shown (Plate C a) which has the
appearance of having taken up several flagella, the ends of
these bodies protruding from the margin of the cell.
The shorter forms bear a strong resemblance to the bacil-
lary bodies observed in the blood corpuscle of cattle suffering
from a chronic or latent form of Piroplasmosis (Plate C a).
This resemblance suggests a possible explanation of these
bacillary forms of Piroplasmosis. In the same blood in
which the flagella were found, the amœboid form of the
Piroplasma and free ring shapes were fairly numerous.
The significance of these flagella is a matter of conjecture.
I have not been able to trace their origin or their function;
Lignière's view that they were simple prolongations of the
Protoplasm of the globules is not probable.
It is very possible that many of the flagellated forms des-
cribed by various authors may be regarded as pseudopodia of
the amœboid stage of the parasite. Several of the illustrations
given by Nuttall and Graham Smith (7) support this view.
The more attractive theory is to regard these free flagella
as mikrogametes similar to the spermoids of the hæmatozoa
of Laveran. Hunt, Doffein, Nuttall and Graham Smith have
considered them from this point of view.
Hunt (8) described crescents in the blood of cattle suffering
from Texas Fever. He observed their change into spheroidal
Piroplasma Bovis.
97
shape comparable to the crescents of the malarial parasite.
Doffein concluded that the large pyriform bodies represent
gametocytes similar to malarial crescents, and that the flagel-
late bodies described by Lignières represent male sexual forms
comparable to those of the Hæmospovidia.
Nuttall and Graham Smith describe a body strikingly
suggestive of a gamete encountered in the blood of a dog
affected with Piroplasmosis.
" These bodies somewhat resemble the crescents of the
human æstivo-autumnal malarial parasite. They are sausage-
shaped, of fairly uniform width and rounded or tapering at
their extremities. Some are markedly vacuolated. In some
the chromatin is almost entirely concentrated in the middle
of the body; in others the particles of chromatin are irregu-
larly distributed."
Similar crescent-shaped bodies were previous to this
observed and described by me.(9) These forms were found
in the blood of cattle in India suffering from a mixed
infection of Piroplasmosis and Trypanosomiasis.
Lingard (10) noted crescent-shaped bodies in connection
with Trypanosomiasis of cattle in India. These cattle were
also the subject of Trypanosomiasis.
Up to the present, no direct proof can be adduced to
establish the identity of these forms with gametes and micro-
gametes. The supposition is based solely on the observation
of forms similar to gametes and microgametes, and on the
analogy of the life cycle of the malaria parasites in man,
birds, batrachia and reptiles.
It is significant that in most instances the flagellate forms
have been encountered in the blood of animals in the last
stage of the disease. In some cases the blood was taken one
or two days before death and in others immediately after
death. The production of these flagellate bodies is, in all
probability, the result of an effort on the part of the parasite
to resist the altered condition of the blood and to prolong
its own existence.
Swingle (11) in a recent article, calls attention to the
similarity, between blood platelets and certain hæmatozoa.
7
98
Piroplasma Bovis.
In Sodium citrate cultures of sheep, rabbit, dog and
human blood, he obtained flagellated blood platelets which in
appearance closely resemble the flagellate stage of Babesia
and some other species.
The platelets in culture show amœboid and flagellate forms
and are capable of movement.
Some of the processes may be regarded as pseudopodia;
the majority are true flagella.
The platelets develop flagella in culture media and also in
the stomach of the tick.
In stained preparations, many forms resemble in nuclear
condition trypanosomes.
The author draws attention to the close similarity of these
flagellated blood platelets and to the flagellate forms of the
Piroplasma Canis, described by Kinoshita, Koch and Kleine.
A comparison of the drawings of Swingle with the illustrations
of the flagellate shapes of the Piroplasma Canis given by Koch
and Kleine show the very striking resemblance. The flagellate
forms described by Nuttall and Graham Smith(12) are also
similar in appearance to the illustrations of flagellated blood
platelets. Swingle concludes that these forms of platelets
observed by him in culture media and in the stomach of the
tick, bear such a marked resemblance in form, size and
structure to Babesia and to the Leishman-Donovan bodies,
that investigators must furnish criteria to differentiate
between the flagellated platelets and the parasites.
Before reading the above-mentioned article, I had been
studying certain forms in the same blood in which the free
flagella of P. Bovis were found.
At the time I believed that these bodies might be connect-
ed with the reproduction of the Piroplasma.
Some circular or oval shapes very much resemble the
"spherical bodies" of the æstivo-autumnal malarial parasites.
These vary in size from half to twice that of a normal blood
corpuscle. They are surrounded by a distinct membrane and
contain numerous chromatin granules. In some cases these
granules are scattered irregularly; in other cells they are arran-
ged parallel to the capsule. Some of these bodies possess
flagella which appear to originate from the chromatin granules,
Piroplasma Bovis.
99
and furnish a close resemblance to microgametes and gameto-
cytes.
Many other forms closely resemble parasites, elongated
bodies with a flagellum at one end and tapering at the other
extremity, simulate trypanosomes; others with oval bodies
possessing flagellum; the free end pointed and containing
fine chromatin granules, behind which a nuclear-like mass
complete the similarity to what has been described as flagel-
lated forms of the Leishman-Donovan bodies. The large
bodies of this kind might well be mistaken for herpetomonas
forms of developing trypanosomes. A very frequent form
observed was that of a spindle with flagella at either end.
While making examination of these flagellated bodies, I had
an opportunity of comparing preparations from the blood of
a patient suffering from a very severe attack of æstivo¬
autumnal fever. In these also flagellated bodies were found
very similar to those met with in the blood of cattle suffer-
ing from Piroplasmosis. These were the same round cells
with membrane chromatin granules and flagella; trypano-
some and herpetomonas forms. Following Swingle's experi-
ments, on culture media, I was able to obtain similar flagellated
bodies from both human and cattle normal blood kept in
Sodium Citrate solution. From these observations it is appar-
ent that many of the flagellate forms seen in the blood of
cattle dying from Piroplasmosis are peculiar developments of
the platelets and in no way connected with piroplasma. This,
however, is not the case with the free flagella described earlier
in this paper, which have no resemblance to any forms of
platelets.
There is the possibility that certain forms of platelets may
have been described as parasites in blood preparations.
Without careful examination such an error might easily
occur. There is, however, no reason to doubt the accuracy of
the observation and descriptions recorded of the flagellated
form of the piroplasma.
PLATE No. C.
Drawn with 2.0 M.M., Apert. 1.30 Zeiss Comp. Oc. 8, Magnification
100, stained Leishman.
A.—Free flagella in blood of cattle suffering from Piroplasmosis Bovis.
100
Piroplasma Bovis.
B.—Forms resembling gametes described by (1) Holmes, (2) Lingard,
(3) Nuttall and Graham Smith.
C.—Flagellated forms of blood platelets in blood of cattle affected
with Piroplasma.
D.—Similar forms of platelets in human blood from a case of æstivo¬
autumnal malaria.
E.—(1) Flagellated forms of Piroplasma Bovis described by Nuttall
and Graham Smith.
(2) Flagellated forms of blood platelets described by Swingle.
REFERENCES.
1. Journal of Hygiene, Vol. IV, 1904.
2. La Piroplasma Bovine, 1903. Arch, de Parasitologie, Vol. VII.
3. Journal of Hygiene, Vol. V.
4. Journal of Hygiene, Vols. IV, V and VI.
5. Generations and Wirtswechsel bei Trypanosoma and Spirochæte.
6. Annales of Tropical Medicine and Parasitology, Vol. II, No. 3.
7. Plate XI, Journal of Hygiene, Vol. VI.
8. Sporozoa, Part I, by Minchin.
9. J. Comp. Path. and Theu., Vol. XVII, Plate VIII.
10. J. Tropical Vety. Science, Vol. II.
11. Journal of Infectious Diseases, Vol. No. I.
12. Journal of Hygiene, Vol. VI, Page 597
PLATE C.
PART VII.
NOTE ON A GIANT POLYNUCLEAR CELL.
ZEITSCHRIFT
FUR HY¬
GIENE, VOL.
LIV.
FOR some years, from time to time I have observed in
preparations of horses' blood large granular cells which I
think has not been previously described. These cells are
usually to be found in the blood of horses in the last stage of
Surra and is so constant a feature in blood preparations taken
at this period, that even in the absence of trypanosomes it is
almost diagnostic of this disease. During the past year, while
examining smear preparations from Bursati sores, the same cells
have been frequently found in large numbers. Some prepara-
tions were almost entirely composed of these large cells, showing
few of the usual microphages and macrophages. In fresh
unstained blood the cell resembles a "mulberry." It is from
two to three times larger than an ordinary polynuclear leuco-
cyte, spherical in shape with the surface studded with large
granules. In stained preparation (Leishman) the mulberry
appearance is still maintained; the granules stain deep red.
These granules appear to be attached to the surface. They
are easily broken off and show beneath the lighter coloured
body of the cell. In many of these cells no nucleus is visible.
The external surface is closely covered with the large deeply
stained granules. In some, where the granules have broken
away, the under surface of the cells appears stained a lighter
red, and at times a light blue. In some preparations a very
distinct lobulated nucleus, stained purple, is to be seen, and
between the granules the under surface stained light blue.
R. Koch describes under the term of "Plasmakugeln" a cell
which from the diagram appears to be somewhat similar to
these " mulberry cells. " These he found in the blood lymph
glands of cattle suffering from East Coast Fever.
ZEITSCHRIFT
HYGIENE UND
INFECKTIONS
Lichtenheld mentions the same Plasmakugeln as being
found only in East Coast Fever.