NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
102
Oesophagostomum radiatum
It should, however, be mentioned that though an abundance of moisture
may restrict the development, the eggs are not easily destroyed under such a
condition.
1-12-39. (i) Normal petri-dish culture in the Laboratory, as control.
(ii) Broken pellets soaked in water in a petri-dish.
2-12-39. (i) First-stage larva in lethargic state.
(ii) Eggs in the segmentation stages.
4-12-39. (i) Second-stage feeding larva.
(ii) A little development observed, but no larva.
5-12-39. (i) Many infective larvæ of O. radiatum in the fæces, with some second-stage
larvæ also.
(ii) Stray second-stage larvæ in the feeding state.. (Fæces getting dry.)
11-12-39. (i) Numerous infective larvæ in water-jacket.
(ii) No larva in water-jacket.
13-12-39. (i) Many infective larvæ in water-jacket.
(ii) Stray infective larvæ in water-jacket.
Till 5-12-39, when the dung was wet and soaking in water, the eggs re-
mained in their segmentation stages, and as the moisture eventually evaporated
and the fæces reached its normal consistency from the 5th onwards, infective
larvæ developed and migrated into the water-jacket as in the healthy culture.
Moisture is of paramount importance for migration of the infective larvæ,
in the soil or elsewhere. When normal petri-dish cultures are left uncovered,
the infective larvae do not emigrate into the surrounding water because the
evaporated water is not allowed to condense on the sides of the petri-dish
with fæces. Nevertheless, when a dish was later placed as a cover over the
same culture, the larvæ were found in the water-belt the next day.
A small dish of distilled water containing infective larvæ was provided
with a belt of water in a larger dish and covered. Daily observations were
made for about a fortnight and no larva was seen in the outer dish. Infective
larvæ, therefore, do not emigrate when they are already in a fluid medium.
Desiccation
Moisture being so essential for the development of the eggs and the migra-
tion of the infective larvæ, the effects of desiccation should be expected to
be highly harmful (by ' desiccation ' it being meant natural drying and not
extraction of moisture by chemical agents). In general, the state of complete
dryness may be very easily reached in the pastures exposed to the summer
temperature, and much less inside cattle-yards and such protected places.
Edges of pools and lakes, the tilled field and the like are considered the most
favourable localities for developing eggs and larvæ where drying is obviated
to a certain degree by virtue of percolating moisture.
Experiment I.—Dung containing eggs of O. radiatum was collected and
distributed as below :—
(a) In a petri-dish, inside the Laboratory (control).
(b) In a mud-pot, at the base of a tuft of grass, moistened and covered
with a glass-bell, inside the Laboratory.
M.
ANANTARAMAN
103
(c) Same as in (b), inside the Laboratory, but not covered. Mois-
tened once a day.
Date
12-8-40 .
15-8-40 .
16-8-40 .
19-8-40 .
22-8-40 .
a
Culture started.
Advanced second-stage
larvæ.
Infective larvæ in faeces;
none in water-jacket.
Infective larvæ in water-
jacket.
Ditto
b
Culture started.
Advanced second -
stage larvæ.
Late second-stage
larvæ.
Sheath
forming.
..
Infective larvæ in
fæces ; none in soil
or grass.
c
Culture started.
50 per cent in second-
stage. 50 per cent in
late first-stage.
No larvæ in dry pellets.
Second-stage larvæ in
moist ones.
..
Infective larvæ and dead
first-and second-stage
larvæ in fæces; none
in soil or grass.
Infective stage is attained by the larvæ in (b) and (c) in a somewhat longer
time than in the control. Because of exposure to the air, larvæ have been
killed in the early stages in dry pellets of (c). Eggs situated in the superficial
area of the dung pellets are more liable to be killed by drying than those in the
interior, unless water was sprinkled on the pellets frequently. No larva was
found to have ascended the grass.
Experiment II.—About 15 infective larvæ were left, on 19 August 1940,
in a watch-glass containing a small quantity of mucus from a person suffering
from cold. The larvæ continued to be active in it in the evening when it was
covered with a petri-dish.
20-8-40.
Mucus dried ; all larvæ coiled and shrunk. Sheath flaccid and body of larva
vacuolated.
21-8-40.
Drops of water added. In 2 seconds, the larval body absorbed the water and
swelled. In 10 minutes, about 50 per cent uncoiled and extended inside
the sheath. In 20 minutes, about 13 extended and more than half of them
were actively moving.
22-8-40.
The water had evaporated. Larvæ coiled and retracted. Water added.
Nearly 80 per cent recovered but took a longer time (about 30 minutes).
23-8-40.
Water evaporated. Some drops of it added. More than 70 per cent revived
in about 45 minutes. Water evaporated by evening.
26-8-40.
Water added. Three of the 15 larvæ were active in about an hour, some slow
and others dead.
This experiment bears evidence to the remarkable capcity of the infective
larvæ to withstand intermittent desiccation, if alternated by wetting. This,
we know, is a condition commonly obtained in nature, the warm or hot day
followed by a cool or dewy night, or even short spells of rain. Moreover,
such complete desiccation as here will be rare, except in summer, since the
larvæ will seek refuge, by retirement, at the bases of grasses, or under the soil
if loose or moist. Even after the lapse of a week, 20 per cent of the larvæ
are normally active and some more just alive. It is noteworthy that the
O
104
Oesophagostomum radiatum
number of larvæ reviving diminishes and the period for reviviscence increases,
as days pass by.
Experiment III.—Nearly 20 larvæ were left dry in a watch-glass at
3-15 P.M. on 29 August 1940. They were coiled and shrunk.
30-8-40.
31-8-40.
2-9-40.
4-9-40.
1-55 P.M.—
2-00 P.M.—
2-15 P.M.—
2-20 P.M.—
2-30 P.M.—
2-40 P.M.—
2-50 P.M.—
3-15 P.M.—
11-30 A.M.—
1-30 P.M.—
11-45 A.M.—
12-30 P.M.—
11-40 A.M.—
12-00 NOON—
Water added.
Uncoiling of most of the larvæ.
Uncoiling not complete.
One larva fully expanded but motionless.
3 fully expanded and moving ; 2 others expanding.
Movement in 6 larvæ, but no locomotion.
9 moving , of which one is very active.
4 active and 6 slow. Others expanded but motionless.
Water added.
4 active, and 4 slow.
Water added.
4 active, and 4 slow.
Water added.
Only 2 slow-moving larvæ.
While 40 per cent of the larvæ were recovered after two days, only 10
per cent were able to revive after six days.
Experiment IV.—The following experiment was conducted to determine
the effect of continuous drying. A small quantity of water containing the
larvæ was poured into a jar with sand and the water allowed to evaporate
completely, and the sand was found to be thoroughly dry on 28 September
1940. On 4 October 1940 a small amount of sand from the top was mois-
tened and a majority of the dried larvæ were discovered to be actively moving
in about half an hour. This reveals the resistence of the larvæ to continuous
desiccation for six days, when in sand. The protectivity of the sand is ob-
vious, as larvæ on a plain glass surface did not revive after drying for two
to three days.
In the act of losing the moisture, the sheath is the first to get affected.
It assumes a crumpled appearance, and the larva coils round itself, and con-
tracting in length and thickness becomes opaque and yellowish. The ab-
sorption of moisture by a dried larva is very rapid and the indications of
revival are an inflation of the body to its original size, and uncoiling, the ex-
pulsion of vacuoles from the intestine, etc. and the re-appearance of the orga-
nization. The uncoiling is in fits, like that of a rusty watch-spring.
Temperature
If any factor can be mentioned as being responsible for keeping down in-
fection with O. radiatum in the tropics, it is temperature, as the influence of
the temperature in those regions is indeed great. Moreover, temperature
does not act alone on the eggs or larvæ ; with increase of temperature, there
is heat, loss of moisture and fermentation in the fæces, each of which by it-
self is an adverse condition for development.
The optimum temperature or the temperature for the normal develop-
ment of the eggs, in Hæmonchus contortus has been indicated as between
20°C. (68°F.) and 35°C. (95°F.) [ Veglia, 1915]. The optimum temperature
for O. radiatum also falls within that range being roughly 75°F. to 90°F. A
temperature moderately higher than this will accelerate the rate of develop-
M.
ANANTARAMAN
105
ment, while 105°F. and over will be beyond the limits of endurance. Cor-
respondingly a lower temperature usually delays development, but excessive
cold will result in the annihilation of eggs or larvæ.
When pellets of fæces are exposed to the sun for a few hours everyday,
the development of eggs in them is more rapid than in the Laboratory cultures.
But, prolonged exposure causes desiccation and death.
Experiment I.—Dung containing eggs of O. radiatum was cultured as
follows:
(a) Pellets broken and spread over moist blotting-paper lining the
bottom of a petri-dish surrounded by a water-jacket (control), inside the
laboratory.
(b) Same as (a), but exposed to sun for three hours each day. The cleavage
and hatching occurred much earlier in (b) than in (a) and the development,
in general, in (b) was quicker than in (a) so long as the exposure did not make
the fæces dry. When the latter condition was attained, most of the eggs and
larvæ were killed. Moistening of the fæces again starts the growth of the
eggs and larvæ which have not been affected by the sun because of their posi-
tion much inside or below the exposed pellets. This experiment was success-
fully attempted in the month of November when the average temperature
ranged about 80°F., but the same if conducted between February and June
with an exposure of even so little as an hour each day will result in the des-
truction of the eggs or larvæ.
Experiment II.—Below are details of an experiment devised to investi-
gate the effects of continuous exposure to the sun, and the possibility of the
infective stages being attained in spite of the sun.
Dung in which eggs of O. radiatum were present was distributed in the
following manner :—
(a) Moistened and placed in a petri-dish surrounded by water, inside
the laboratory (control).
(b) placed in a plot of ground and directly exposed to the sun, and
moistened occasionally, and
(c) placed as in (b), but was covered with a layer of loose sandy soil
and moistened occasionally.
Date
15-1-40 .
16-1-40 .
18-1-40 .
19-1-40 .
20-1-40 .
a
Culture started . .
Hatched
first-stage
larvæ.
Late second-stage larvæ.
Infective larvæ in water-
jacket.
More infective larvæ .
b
Culture started . .
Eggs still in cleavage
stages.
Dead first-stage larvæ.
All dead larvæ . .
Ditto.
c
Culture started.
Hatched first-stage larvæ.
Early second-stage larvæ.
Late second-stage larvæ.
Infective larvæ in deep
pellets.
Average temperature 90° F.
The progressive development in each case is tabulated above and pre-
cisely at the end of the 4th day, the first few infective larvæ migrated into the
water-jacket in the control culture (a). At that time, only dead first-stage
larvæ were discovered in (b). On the contrary, in (c), though the larvæ had
C 2
M.
ANANTARAMAN
107
the metal is placed on the watch glass for sometime and the heat conducted
to the water. Their activity is increased when the water is slightly warm,
but when moved over a flame or exposed to the sun for long they die out-
stretched.
Light
Dull light, as exists in a cloudy weather and at twilight, or darkness is
the most conducive to the normal development of the eggs. Bright light,
to the degree of being a glare, results in delayed cleavage and hatching and
sometimes in the death of the first-stage larvæ. The effect of direct sunlight
cannot be said to be exclusive, since a higher temperature and absence of
moisture exert their influence collateral with the sun's light. Growth progress-
es when there is minimization of direct sunlight by covering the fæcal mass
with loose soil or by leaving the pellets under a hedge where shade is
continuous.
Toxic substances
It frequently happens that consequent to some disturbance in the host,
there are present toxins in its fæces which have an untoward influence on the
eggs imbedded in the dung. Foul-smelling, diarrhœic or fermenting fæces
are not favourable to development. In artificial cultures fermentation was
prevented by the addition of charcoal powder to the fæces.
Various chemical substances have been testified to be injurious
to the eggs and larvæ. Bouin's fluid, picric acid, formalin or corrosive
sublimate kill the eggs and cause the infective larvæ to coil on themselves
immediately on contact, and to be killed soon after.
Since the larvæ withstand acidity in the stomach of the host an investiga-
tion was made to calculate the maximum strength of it that would be innocu-
ous to them. A series of dishes containing water with hydrochloric acid added
in the proportion of ½, 1, 2, 2½, 3 and 3½ per cent was arranged and a number
of infective larvæ placed in each. The larvæ were discovered to be living
in the first three even 24 hours after, to tolerate the 2½ per cent solution from
four to five hours, the 3 per cent solution for about an hour, and to be dead
in the 3½ per cent solution immediately after.
A number of free-living nematodes young and adult, recovered from the
soil and moist fæces by the Baermann's method, were mixed with an almost
equal number of infective larvæ of O. radiatum, and left in water containing
(i) 1 per cent HCl and (ii) 2 per cent HCl. It was found, on examination
after five minutes, that while all the infective larvæ continued to swim un-
affected, the free-living nematodes were dead even in the 1 per cent solution
and disintegrating.
Solutions of the disinfectant Izal in water were prepared in the proportion
of (a) 1 in 10,000 (b) 2 in 10,000 and (c) 4 in 10,000. A small quantity of each
of these was taken in a petri-dish, on 20 November 1940 and a number of
infective larvæ were left in each at 12-30 P.M. Microscopic examination
every hour, till 4-30 P.M., revealed no change in the activity of the larvæ. On
21 November 1940 and 22 November 1940, the larvæ in all the three con-
tinued to be active. It was therefore concluded that a solution of Izal in
108
Oesophagostomum radiatum
water, in any of the above concentrations, will not have a detrimental effect
on them.
The same experiment was repeated on 25 November 1940, with higher
concentrations, (a) 0.5 in 1,000 (6) 1 in 1,000 and (c) 2 in 1,000. The observa-
tions were:—
25-11-40.
26-11-40
1-55 P.M.—
2-45 P.M.—
3-30 P.M.—
11-00 A.M.—
Experiment started, with- infective larvæ in each.
(a) 40 per cent motionless.
(b) 70 per cent and
(c) 80 per cent.
(a) 40 per cent motionless.
(b) 70 per cent and
(c) 80 per cent.
(a) 50 per cent active, others extended and motionless.
(b) 80 per cent extended and inactive, 20 per cent of the total
disorganized.
(c) All extended and motionless, 50 per cent dead.
In 22 hours, a solution of 1 in 1000 destroys 20 per cent of the larvæ
and that of 2 in 1000, 50 per cent. It may be judged from this that the latter
may kill the entire larval population in 30 to 35 hours.
Copper sulphate was the next to be tried. Solutions of this in water to
make up concentrations of 0 . 5 per cent and 1 per cent were made and a few
infective larvæ left in three petri-dishes containing (a) water, (b) 0.5 per cent
solution and (c) 1 per cent solution.
29-11-40.
30-11-40.
11-15 A.M.—Experiment started.
12-00 Noon.—
2-15 P.M.—
4-00 P.M.—
10-30 A.M.—
(a) All larvæ active (control)
(b) 15 per cent extended and dead, 10 per cent motionless.
(c) 30 per cent extended and dead, 10 per cent motionless.
(a) All active.
(b) 20 per cent dead.
(c) 50 per cent dead.
(a) All active.
(b) 50 per cent dead.
(c) 70 per cent dead.
(a) All active
(b) and (c) All dead.
In reviewing the rate at which death begins in the larvæ, the 0.5 per cent
solution would appear to be enough to kill all the larvæ in about 10 hours, and
the 1 per cent solution in six to seven hours.
Dung containing eggs of O. radiatum was divided into equal quantities of
20 grams each and cultured as follows, in petri-dishes with water-jackets, on
7 January 1941. To each fæcal mass were added 5 c.c. of the following
solutions in water:
(a) Distilled water (control)
(b) Izal—2 in 1000.
(c) Copper sulphate—0.5 per cent.
(d) Lactophenol—1 per cent.
(e) Water in which neem leaves were crushed.
The development proceeded with almost equal rapidity in them, and on
12 January 1941, infective larvæ were found in the water-jackets of all of
them.
M.
ANANTARAMAN
109
Though Izal and copper sulphate in the proportions mentioned above
were found to destroy the larvæ when they were directly placed in them, the
foregoing experiment proves how ineffective they are when mixed with the
fæces. The ostensible reason is that the chances of the larvæ coming in
contact with the solutions are reduced, due either to the insufficiency of the
solutions or to the solutions percolating down rapidly leaving the upper
pellets of the fæces unaffected.
Assuming that 10 c.c. of the above solutions for every 20 grams of fæces
would be a fair proportion, a similar experiment was started on 8 January
1941. On the. 13th, infective larvæ were present in all the water-jackets
except that of (d) in which lactophenol was employed, but on the 14th a very
small number of them appeared in (d) also. Thus, none but lactophenol has
any effect when mixed with the fæces.
Twenty-five grams of the same fæces were mixed with the following and
cultured, on 18 February 1941.
(a) 5 c.c. distilled water.
(b) 5 c.c. 3 per cent hydrochloric acid.
(c) 5 c.c. 3 per cent sodium hydroxide.
(d) 10 c.c. 3 per cent hydrochloric acid.
(e) 10 c.c. 3 per cent sodium hydroxide.
(f) 5 c.c. 2 per cent formalin.
On 25 February 1941, infective larvæ were observed in all except (e)
and (f). It was also noted, in passing, that in no culture was any fungus
found to have grown.
Five cultures of the dung were made in this manner on 17 January
1941 :—
(a) usual culture, inside the room.
(b) plus 10 c.c. of urine from a cow, inside the room.
(c) usual culture as in (a), inside an incubator at 37 degrees C.
(d) plus 10 c.c. of urine from a cow, inside the same incubator.
(e) plus 10 c.c. of urine from a cow, in the open field.
22 January 1941.
(a) Infective larvæ in water-jacket.
(b)
(c) }No larvæ in fæces or water-jacket.
(d)
(e) No larvæ in fæces or soil.
While urine alone is found to be harmful to the eggs in (b) it is not sur-
prising that the eggs should have been destroyed in (d) and (e) where there
was a higher temperature and consequent desiccation.
Lime powder is well known as an effective poison for infective larvæ and
has been successfully applied on open spaces to destroy infective larvæ of
O. radiatum. That it should be immensely useful in the direction of disinfect-
ing pastures and limited areas will be no exaggeration.
M.
ANANTARAMAN
111
gible extent and, as would be seen from an experiment described below, the
larvæ confine themselves to the soil-surface if there is even a film of water.
Dispersal could therefore be made possible by water running on the pastures
and fields.
Fæcal pellets placed on soil and moistened frequently show infective
larvæ as usual, which sooner or later migrate into the soil right below the
fæces. Such larvæ have been noticed to burrow into the soil, exposed to the
sun, etc. to a depth of 2 inches (5 cm.) as in the case of the infective larvæ
of Hæmonchus contortus [Veglia, 1915].
Experiment I.—On 26 March 1941, the height of two rectangular jars
was graduated in centimetres, from the bottom, and (a) one was filled with
coarse sand, and (b) the other with soft mud. The sand and mud were soaking
in water which stood about a centimetre over them. Numerous infective
larvæ were gently pipetted into the water in each, and the jars were covered
to prevent evaporation, and disturbance by breeze, etc.
29 March 1941.—The supernatant water in both contained many larvæ.
(a) Larvæ were found up to a depth of 3 cm.
(b) Larvæ were found up to a depth of 2½ cm.
31 March 1941.—Numerous larvæ in water.
(a) Larvæ, up to 3 cm.
(b) Larvæ, up to 3½ cm.
The extent to which the larvæ have migrated down here is much less than
in normal soil where water is not present over the soil abundantly. The
presence of the larvæ in the supernatant water suggests that in small ponds
and pools in the field, they may not once for all settle on the bottom, but keep
on rising to the surface after periodical rest, so that water impregnated with
them will be taken in by the cattle. Disturbance of the water by breeze or
the cattle themselves will mean definite facilities for the ascent of the larvæ.
Experiment II.—Two sets, of five test-tubes each, one for sand and the
other for mud, were arranged. A large number of infective larvæ were placed
at the bottom of each tube in a small quantity of water, and sand or mud gently
dropped over to the heights of 6, 7, 8, 9 and 10 cm. on 26 March 1941.
Clean tap-water was poured into the tubes by drops till the soil was soaked and
some more water stood above it.
The supernatant water examined on the following two days revealed the
following :—
27-8-41
29-3-41
Sand ....
Mud
.
.
.
.
Sand ....
Mud
.
.
.
.
6 cm.
2 larvæ
3 larvæ
3 larvæ
30 larvæ
7 cm.
1 larva
8 larvæ
3 larvæ
35 larvæ
8 cm.
Nil.
7 larvæ
4 larvæ
7 larvæ
9 cm.
1 larva
Nil.
Nil.
6 larvæ
10 cm.
Nil.
20 larvæ
1 larva
10 larvæ
The larvæ seem to traverse a height of even 10 cm. to reach the surface
of the earth where they can be swallowed by the animals, provided moisture
is available throughout. This, in contrast to their penetration down the soil
to about 3 cm. should be regarded as negative geotropism. The presence of
larvæ in large numbers over the mud is attributable to the softness of the mud-