S. K. SEN AND F. C.
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151
separate cultural examination of the flies' heads
and bodies was made, the heads having been
removed immediately after the flies were killed.
The wings were also removed. It was advan-
tageous to ' clean ' the material in the following
way: it was first well shaken up in ten changes
of sterile distilled water, drained as much as pos-
sible, immersed in absolute alcohol for five to ten
minutes with occasional shaking, and dried on
absorbent paper. The dried material was then
powdered in a mortar and suspended in saline
(2.5—10.0 c.c.) from which serial tenfold dilu-
tions were made for plating on agar. Faecal
spots, desposited by the flies on Petri dishes,
were likewise suspended in 2.5—5.0 c.c. saline.
After 24 hours at 37°C. anthrax colonies can be
recognized at a glance if they are numerous, and
there is little contamination. They can still
be recognized with fair certainty, even if there
are considerable numbers of discretely-growing
contaminants. When there is extensive conta-
mination of the spreading type, the anthrax
growth is masked. When necessary, colonies
are picked and injected to white mice for
identification.
The numbers of anthrax bacilli in the bodies of
Stomoxys flies after feeding on the infected goats
varied considerably. In 12 instances, the numbers
of colonies estimated to have developed from a
single fly ranged from 1,250 to 1.2 million;
in 10 of the 12 instances, the range was from
15,000 to 142,000.
TRANSMISSION EXPERIMENTS
I. Experiments with Stomoxys calcitrans
1. Feeding infected flies on normal skin (Table
1). The flies were fed on anthrax-infected goats
for varying lengths of time and then fed on
healthy goats after intervals ranging from 0 to
48 hours. The main object of these experiments
was to test the possibility of the disease being
transmitted by the so-called ' interrupted '
method of feeding, i.e. by interrupting the flies
while engaged in feeding on the donor and allowing
them to complete their feed on the receptor. In
each case, the feeding on the donor was done only
when anthrax organisms were actually present in
the circulating blood. (The time elapsing between
the finding of anthrax bacilli in the blood and
the death of the animal was frequently five to
eight hours or even more, so there was plenty of
time for the flies to feed). In a few instances,
the flies were allowed to feed completely on the
donor, and in others, carcases were used as
donors. Mitzmain [1913, 1] has given 2½ minutes
as the average length of time taken by S. calcitrans
to obtain a full meal of blood from goats. Expe-
rience at Mukteswar has shown that the duration
of feed of individual flies is very variable, depend-
ing on the condition of the goat and of the fly
itself and also on the temperature of the at-
mosphere. When the flies are being fed en masse,
some allowance has also to be made for the fact
that all the flies of a lot will not settle on the host
at the same time, whilst individual flies are liable
to be interrupted in the feed by the movements
of other flies of the lot.
2. Feeding infected flies on scarified or cauterized
skin (Table II). The object of these experiments
was to test the possibility of the disease being
produced by infected flies biting on fresh wounds
or abrasions on animals. The flies were confined
in ' scarification cages ', fed on incisions made
with a scalpel on the flanks of goats dead of
anthrax and then transferred to the cauterized
skin of healthy goats. The incisions were about
three inches in length and one inch in depth.
The cauterization was done by applying a heated
spatula to the skin of the animal and then remov-
ing the epidermis of the scalded area by the blade
of a scalpel, care being taken to avoid bleeding
in the process. In the earlier experiments,
the skin was scarified by making a series of super-
ficial cuts on it by means of a scalpel, but this did
not prove satisfactory owing to bleeding and
hardening of the scarified area that followed.
3. Causing infected flies to defaecate on scarified
or cauterized skin (Table III). In some instances
faeces of infected flies, when tested culturally,
showed the presence of anthrax organisms.
This led to a series of experiments to find out if
under natural conditions defaecation by Stomoxys
on abrasions or wounds was likely to produce the
disease. For the purpose of these experiments,
the infected flies were confined in ' scarification
cages' and brought in contact with the cauterized
skin of healthy goats. At the end of the feeding
period, some dozens of faecal spots could be
counted on the exposed area of the skin. The
results were negative.
In connection with these experiments, it was
demonstrated that, as expected, anthrax can be
set up in goats by lightly applying culture suspen-
sion to the freshly-cauterized skins, but not to
skin from which hair has been carefully removed
by scissors. Also, as expected, infection is
produced when culture suspension is lightly
applied to an area of skin, after removal
of hair by scissors and after hungry Stomoxys
have been allowed to feed on the area for 30
minutes or so. The bites rapidly cause an
oedematous thickening of the skin, with bleeding
points.
II. Experiments with Musca domestica
1. Bringing infected flies in contact with cau-
terized skin (Table II). These experiments were