NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
84
ABSTRACTS
received a second dose of the vole strain. One calf died of allergic reaction following
the inoculation of the second dose. Another calf died from non-specific causes. All
the rest remained apparently healthy. Two of these were killed 44 and 89 days respective-
ly after the injection to follow the reaction due to the inoculation of the vole strain of
acid-fast bacilli. From careful post mortem, histological and cultural examinations, it
was concluded that the vole strain was able to excite in calf a tuberculous reaction,
chronic in nature, which tended to disappear without leaving many traces. It was also
ascertained that sensitiveness to tuberculin following injection of the vole strain deve-
loped very early.
(2) Immunity resulting from vaccination with the vole strain.—The remaining nine
calves (four once-vaccinated and five twice-vaccinated) were tested for their resistance
to tuberculosis along with two controls by the oral administration of 7.5 mg. of viru-
lent bovine tubercle bacilli. The interval between vaccination and resistance tests rang-
ed from 65 to 175 days. The calves were killed at periods ranging from 208 to 223 days
after the resistance tests. Careful post mortem examinations were made and selected
glands were tested for infectivity by guinea-pig inoculation.
Both controls became infected, the glands of the alimentary tract showing wide
spread lesions and some dissemination, slight in one, a little more extensive in the other.
Of the nine vaccinated calves, five were found to have trivial tuberculous lesions in
the glands adjoining the alimentary tract and four showed no macroscopic lesions. Select-
ed glands from the five calves with trivial lesions were tested for evidence of viable orga-
nisms, two guinea-pigs being used for inoculation with material from each calf. In most
instances only one of the two inoculated guinea-pigs developed the disease, showing that
living tubercle bacilli were very scanty in the glands. Of the four no-lesion cases, guinea-
pig inoculation revealed the presence of living bovine bacilli in the mesenteric glands of
two ; the other two harboured no living bovine bacilli.
Of the four no lesion calves, three had received a single dose of the vaccine, (2 intra-
venous and one subcutaneous) and one two doses of the vaccine. It therefore appears
that there was no advantage in giving more than one immunizing dose. As regards
route of administration, the intravenous route is preferred to the subcutaneous, as in
the former no unsightly local reaction was obtained.
As the authors conclude, the results obtained in calves by vaccination with the
vole strain of acid-fast bacilli were good and the method is worth further trials. [V. R. R.]
REVIEW
Diseases transmitted from Animals to Man. Second edition. By THOMAS
G. HULL, PH.D. (Charles C. Thomas, Springfield, Illinois, U. S. A.,
1941, pp. xiii and 403, 45 illus. cloth, $5.5)
F OR a variety of purposes animals have been brought under man's care and
dominion. They have been the motive power for his progress from ' cave¬
shelter to pent-house. ' They have supplied him both ' bountiful larder and
plentiful labour. ' But this ' deviation of man from the state in which he
was placed by nature seems to have proved to him a prolific source of disease.'
Animals have not only ministered to the wants of man and even saved millions
from death but ' they have also sent untold millions to death. ' Diseases of
animals are therefore of great importance in relation to public health.
A review of current literature shows that the role which animals play in the
dissemination of disease among human beings has become the acute problem
of the day and the appearance of the second edition of Hull's book at this time
is really very opportune. The object of the author as stated in the preface
to the first edition has been to afford a common meeting ground where the
veterinarian, the physician, the laboratorian and the health official might under-
stand each other's phase and angle of view of the problem of diseases trans-
missible from animal to man, which so immediately concerns them and through
their concerted efforts to reduce the number of infections which man contracts
from animals.
The first edition of this book appeared in 1930. In this second edition
the author, as stated in the preface, has revised and brought up-to-date
all chapters, re-written those on animal parasites, fungous diseases and psitta-
cosis and added new chapters on louping ill, sore mouth of sheep, equine
encephalomyelitis, Rift Valley fever, typhus fever and relapsing fever. This
shows what a vast amount of advance has been made in the short priod of 11
years. Having been deprived of full opportunities for personal observations
in connection with these advances the author has sought the aid of workers—
all experts in their particular fields—in the preparation of his book. His choice
so far as America is concerned, has been good. A better team of collaborators
would be hard to find, consisting as it does of expert physicians, veterinarians,
laboratorians and health officials. The names of famous workers like Huddle-
son on brucellosis, Kelser on rabies, Meyer on psittacosis and equine encephalo-
myelitis and Mohler on tuberculosis and Tanner on botulism are sufficient to
underwrite the authenticity of the text. We are assured that we are getting
information at first hand and this lends great authority to the work.
The book is divided into five sections. Section I contains seventeen chap-
ters and deals with different diseases of domesticated animals and birds, viz.
tuberculosis, anthrax, foot and mouth disease, brucellosis, milk sickness, small
pox and cow-pox, glanders, rabies, psittacosis, swine erysipelas, louping ill,
sore mouth in sheep, equine encephalomyelitis, Rift Valley fever, food
infections due to Salmonella group, infections produced by animal parasites,
85
ORIGINAL ARTICLES
THE LIFE-HISTORY OF OESOPHAGOSTOMUM RADIATUM,
THE BOVINE NODULAR WORM
BY
M. ANANTARAMAN, M.A., M.Sc.
Madras Helminthiasis Scheme, Madras Veterinary College
(Received for publication on 11 July 1941)
(With Plates IX-XI and 25 text-figures)
INTRODUCTION
T HE Imperial Council of Agricultural Research, in inaugurating a Helmin¬
thological Research Scheme in the Madras Presidency, has chosen definite
parasites of cattle for a study of their life-histories, so that the data col-
lected therein would form the scientific basis for the suggestion of control
measures. This article, representing the results of a work which extended
over nearly two years of experimentation, is the first of a series of such studies
and deals with the ubiquitous and harmful nodular worm, Oesophagostomum.
This parasite is also important from an economic viewpoint in respect of
emaciating the cattle or making their intestines nodular and unfit for sausages.
The need, therefore, for a study of this nature has been indeed great.
HISTORICAL
Oesophagostomes have been known to attack man, monkeys, camel,
pig, cattle, sheep and goats, practically all over the earth, and are of the follow-
ing species : O. dentatum (Rudolphi 1803 ), O. longicaudum ( Goodey 1925 ),
O. apiostomum (Willach 1891), O. brumpti (Railliet and Henry 1905),
O. stephanostomum ( Stossich 1904), O. columbianum (Curtice 1890 ), 0. venu¬
losum ( Rudolphi 1809 ), O. asperum ( Railliet and Henry 1913 ), O. radiatum
( Rudolphi 1803 ) and O. multifoliatum ( 1933).
On making a general survey of the life-histories of Oesophagostomes,
it is found that Oesophagostomum apiostomum of man and monkeys was studied
by Walker in 1913. Weinberg [ 1908] and Thomas [ 1910 ] observed that
the larva was carried by blood to the wall of the small intestine, where it
escaped from the blood-vessel, encysted in the sub-mucosa, in the internal
or external muscularis, or in the sub-peritoneal space, and there grew into the
87
90
Oesophagostomum radiatum
now not to happen. Whether the larva in the South African cattle was that of
O. inflatum or O. radiatum he could not decide, but inclines to the view that
it may be the latter. He regards that the disease is not as grave in Africa
as in France, since many cattle slaughtered in the Cape had innumerable
nodules in the intestines though having been seemingly healthy before death.
This is attributed to the variation in the intensity of the infection or the degree
of resistance of the host.
Bontz and Krause [ 1930], according to Monnig [1933], mentioned intus-
susception as one of the effects of the nodular verminosis in bovines, with
which Monnig disagrees, and concluded that the causal larvæ were those of
Bunostomum in view of the occurrence in them of a large buccal capsule with
teeth at the base and an anteriorly situated vulva. To disillusion this, Monnig
obtained larvæ from similar nodules and after a comparison with the larvæ
of O. columbianum in sheep [ Veglia, 1923] placed the former under Oesphagos¬
tomum, and even suggested the species as O. radiatum. He explained that
the vulva noticed by them behind the œsophagus may have been an artefact
and the coiled uterine tubes ' the necks of the cervical glands '.
MATERIALS AND METHODS
For the collection of eggs of O. radiatum, females of the worm were ob-
tained from cattle in the Madras Cattle Slaughter House. When the contents
of the colon are washed, the worms would wriggle out of the fæcal mass. With
a little experience they could be identified with the naked eye and transferred
to the laboratory in normal saline. The dung of the animal which harboured
these worms very often contained the eggs in large numbers and was then
put to culture. On teasing a gravid specimen also with a fine mounted needle,
numerous eggs were released from the uterus. When, either by experimental
or natural infection, a donor-calf was procured, the eggs appeared in abun-
dance till the death of the host.
As far as possible, fæces laden with eggs was taken direct from the rectum
of the host or when it was being passed out, so that fresh and uncontaminated
material was used. Such fæces was broken up slightly, and moistened if
dry or hard, or mixed with sterile dung if thin or diarrhoeic, before being sub-
jected to culture. The cultivation of larvæ from the eggs was successfully
achieved by more than one method.
(1) The glass-jar method
A tall, rectangular glass-jar is lined on the inside with moistened blotting
paper, and fæces with the eggs of the worm is evenly and loosely laid over the
bottom. The mouth of the jar is covered with a lid and the jar placed in dull
light in a room. After the lapse of from four to six days, the infective larvæ
climb up the sides of the jar in colonies and can be collected by either washing
the paper in water or removing them with a wet camel-hair brush.
M.
ANANTARAMAN
91
(2) The petri-dish method
This has proved to be better than the glass-jar method, in being handy
and in respect of the collection of larvæ being easier and in clean water. Two
large petri-dishes of the same size and a third smaller one are necessary. The
bottom of the small dish is lined with damp blotting paper and the broken
fæces spread on it. This is then placed in one of the large dishes and the
intermediate space filled with distilled water. The other dish is placed over
these to prevent evaporation, and the entire apparatus placed in a cool por-
tion of the room. Under ideal conditions, the infective larvæ scale over the
edge of the small dish at the end of the fourth day and migrate into the water-
jacket. All the larvæ can then be pipetted off this dish into another for
examination.
(3) Distilled water
Fertilized eggs of O. radiatum were obtained from dung or by opening
gravid females, and when left in this medium to develop segmented and
hatched into the first-stage larvæ in nearly 20 hours. The larvæ lived for a
while and died for want of food.
(4) An emulsion of fæces
Cleavage and hatching were accomplished and development up to the
early second stage was possible, as food was available. But further growth
was arrested since the first ecdysis could not be undergone in a fluid medium.
(5) Agar inoculated with B. coli
Since the agar had to be of a viscous consistency, 20 per cent agar was
dissolved in three parts of water and autoclaved. A loop of B. coli was sown
into the medium and the agar incubated for at least six hours. First-stage
larvæ hatched in distilled water or fæcal emulsion, or eggs from female
worms or dung were placed in a watch-glass containing this agar and covered.
Normal development followed and infective larvæ were observed at the end
of the fourth day. The bacteria serve as food, and ecdysis is possible by
reason of the viscosity of the medium. Microscopic examination is facilitated
as the activity of the larvæ is restricted in this.
The preparation of the drench.—The petri-dish method proved to be the
most convenient for the cultivation of infective larvæ and was therefore
largely employed in this work. The infective larvæ of O. radiatum have a
fine, whip-like tail and could be identified with a magnification of 20 times.
The petri-dish of water containing the larvæ was placed on the stage of the
binocular microscope and with a pipette, whose tip was finely drawn and bent
at an angle, the larvæ were picked one by one and counted alongside, if neces-
sary. The pipette was emptied into a feeding-cup with a little water and the
suspension of the larvæ administered orally to the experimental animal.
92
Oesophagostomum radiatum
Care of the host.—The subjects for experiments were all calves varying
from three months to a year in age, at the time of purchase, of both sexes and
species—B.indicus and B. bubulus. The dung of every calf was examined by
both the centrifugalization and floatation methods to ensure accuracy, as far
as possible, for nearly a month before an experiment on the animal was com-
menced. As it was occasionally found in practice that eggs escaped detection
on account of their scantiness, the dung was also cultured in petri-dishes and
the water-jacket searched for any infective larva. Routine examinations of
fæce, and records of weight and temperature were made regularly during the
experiments and post-mortem, examinations conducted as soon after death as
possible. During autopsy, each section of the alimentary canal, as the small
intestines, colon, etc. was closed at both ends by tying a piece of thread and
then only severed, to avoid mixture of the contents. The intestinal con-
tents were diluted with water and allowed to stand, when the sediment was
searched for parasitic stages of the worms. Nodules were cut and transferred
to the laboratory in normal saline and examined for larvæ. When a large
number of such larvæ were required, the nodules were left in the medium in
an ice-chest and dissected even till the third day after autopsy.
All tissues were normally fixed in 5 per cent formalin, while for nodules
to be sectioned Bouin's fluid proved satisfactory. After fixation in this for
from 10 to 15 hours, the tissues were repeatedly washed in 70 per cent alcohol,
in which a small quantity of lithium carbonate served to remove the picric
acid. Paraffin of a melting point of 58°C. was employed, sections were cut to
a thickness of 4 to 6 microns and stained in Delafield's or Ehrlich's hæmatoxy-
lin and eosin. The intra-nodular larva may be observed in its natural posi-
tion, if a nodule is sectioned horizontally and delicately with an ordinary
razor and all the pieces so obtained transferred to carbolic acid from 70
per cent alcohol. In a very short time, the slices are rendered transparent
and on examination in a binocular microscope, the larva would appear as
a greyish curve in one or two of the sections. Permanent slides have been
obtained by mounting the required piece in carbolic acid on a cavity slide
and sealing the edge of the coverslip with Canada Balsam. For examination
of the infective larvæ, Gram's Iodine serves best by killing them instanta-
neously and straightening them, whereas in formalin, Bouin's fluid or corrosive
sublimate they live for some time in a coiled state.
THE EGG AND FREE-LIVING STAGES
The egg
The ova are formed round the central core of each of the two ovaries and
are liberated into the uteri. All stages in the maturation of an ovum can be
followed by opening a mature female worm. The oocyte is formed after the
separation of the two polar-bodies which may be usually seen at a pole of
the ovum and attached to the yolk (Fig. 1). Fertilization is effected as a
result of copulation and every pregnant female may be identified, without
M.
ANANTARAMAN
93
mistake, by the presence of a dark sticky mass at the vulval area, which
perhaps serves to give a stable attachment between the pair.
FIG. 1. An unfertilized egg with polar bodies.
„
2. A fertilized egg in unicellular condition.
„
3. Egg after first cleavage, with two identical cells.
„
4. Egg after second cleavage, with four cells.
„
5. Morula stage.
„
6. Tadpole stage with broad anterior end and a blunt narrower posterior region.
„
7. Embryo taking the shape of a worm. Tail being differentiated.
„
8. Fully developed vermiform embryo with mouth, œsophagus, intestine and fine tall.
The fertilized egg is ellipsoidal with somewhat flattened sides, possesses
a smooth, transparent shell about 1 micron thick, and measures nearly 950
by 550 microns. The yolk is granular and faintly yellowish and does not fill
the cavity of the shell near the poles. The nucleus is recognized as a clear
spherical area, ex-centrically placed and 22 microns in diameter (Fig. 2).
In its passage along the uterus to the vulva, the egg cleaves twice ; the first
cleavage is equatorial and the two daughter-cells are flattened in the plane
of contact (Fig. 3), the second is meridional and produces four blastomeres
which shift and so arrange themselves that there are two cells in the equa-
torial plane close to each other, one on top and the other below (Fig. 4).
In this condition the eggs escape out into the intestine of the host. Such
eggs have been mechanically expressed out of the female and allowed to
pursue their development in distilled water, emulsion of fæces, or agar.
M.
ANANTARAMAN
95
0.015 to 0.02 mm.; the tip of the tail is 0.12 mm. away from the anus.
The cuticle or skin is thin, transversely striated and projects on the sides from
the oephalic region to a little posterior to the anus as the lateral lines which
bear a sharp edge.
FIG. 9. First-stage larva, soon after hatching, showing attenuated tail, rhabditoid
œsophagus, chyle intestine composed of 16 cells, nerve-ring, excretory canal
and gential primordium between the 4th and 5th cells.
98
Oesophagostomum radiatum
35 hours after the first ecdysis or about the close of the third day of develop-
ment. The features of the slumbering larva are that a fresh skin is being
separated all round the body, the buccal cavity is narrowed to a slender line,
the œsophagus loses its rhabditoid shape, the valvular junction fades away
FIG. 10. Second-stage larva, still with slender tail, somewhat rhabditoid œso¬
phagus, intestine of 18 cells, genital primordium between 5th and 6th cells, etc.
M.
ANANTARAMAN
99
and the intestinal cells become indefinite in outline. The larva enters the
third stage thereafter.
The third-stage larva
Reviving from the quiescence, the larva attains the third free-swimming
stage, when a moulting has occurred but no ecdysis. The old skin is retained
FIG. 11. Third-stage or infective larva. Strongyliform œsophagus, 20 cells
making up the intestine, genital rudiment between 6th and 7th cells, a
cuticular design near the mouth, a long tail and a loose sheath.
M.
ANANTARAMAN
101
Repeated observations on the infective larvæ collected and stocked in
bottles of distilled water revealed that the larvæ were gaining a transparency,
after sometime, due to the expenditure of the reserve food-granules in the in-
testinal cells. To determine whether this was caused by the absence of food
in the distilled water or by the cessation of the feeding activity of the larvæ,
the following experiment was conducted.
12-2-40
13-2-40
to
16-2-40
Larvæ collected about 15-1-40 in distilled water, and very transparent, were
picked and transferred to (i) a watch glass containing fæcal emulsion, and
(ii) another containing agar with B. coli.
In both, larvæ were still transparent, and no appearance of food-granules in
the intestine was noticed.
In spite of the fact that food-material was made available, the larvæ
did not show any indication of having fed on it. Morevover, the closed nature
of the mouth and anus definitely proves that feeding is suspended during this
stage.
THE ENVIRONMENTAL INFLUENCE
Air
The presence of air is one of the chief requirements for the successful
development of eggs and larvæ. This fact is evident when eggs fail to flourish
if air is expelled in a culture by compressing the dung into a cake or by making
a paste of it with water. The quantity of air necessary appears to be very
small as development is not retarded when small tubes, petri-dishes or bottles
containing the fæcal masses remain tightly closed. In support of this argument
is the fact that the cleavage of the egg from the four-celled stage in which it
is laid by the female in the large intestine of the host to the multicellular stage,
proceeds uninterruptedly in the rectum where the supply of air cannot be large.
Yet, further development into the larva will not be observed in fæces unduly
retained in the rectum as, then, the toxicity caused by the constipation will
prove to be injurious to the eggs. It is also not unlikely that an aeration of
eggs to a greater degree than the minimum will accelerate the rate of their
development. So, this may be offered as an explanation for the unequal
development noticed in the different regions of the fæcal pellets.
Moisture
For normal cleavage and larvation, the fæces should be moist, as much
as it would be when evacuated by a healthy host, and this condition is fulfilled,
in the laboratory cultures by placing a damped filter-paper below the dung
and preventing vaporization, and in the field probably by rain or dew. When
fæcal pellets thrown in the open were not moistened frequently, eggs were
noticed to be killed.
An excess of water delays or stops growth. When the fæces is left soaking
in water or mixed with water, the eggs do not hatch, or the hatched larvæ
are killed soon after. This is the result of the expulsion of the air by the
water and the consequent denseness of fæcal particles. No development
was observed in tubes in which the dung was immersed in water.