192
Bovine on Pleuropneumonia
tube. Considerable initial difficulty was experienced in obtaining surface cultures of the Assam
pleuropneumonia virus on Bennett's serum agar until the simple technique of Wright [1933] was
followed, viz. extracting the minced heart muscle and peptone together at 5°C. for 24-48 hours instead
of preparing the extract first and adding the peptone later. On the other hand, we had no difficulty
in obtaining primary suface cultures of the Australian strains on solid medium prepared according
to Bennett's original formula. The choice of the serum used in the preparation of the medium is,
in our experience, a matter of some importance. The serum of Kumaoni cattle is unsatisfactory in
comparison with horse serum; growth of the Assam organism is relatively poor and primary isola-
tion from Berkefeld V filtrates frequently fails in Bennett's medium containing cattle serum. Austra-
lian strains, however, grow in the medium equally well whether it contains cattle or horse serum.
It has also been our experience that all samples of horse serum, are not equally conducive to growth
of the Assam organism; one sample seemed definitely inhibitory, one was of mediocre value and only
one of three samples tested was really satisfactory. B.V.F.O.S. medium, a special culture medium
favoured by Australian workers [Turner, Campbell and Dick, 1935] gave poorer results than Bennett's
medium. Our experience of the medium was, however, limited to one batch (difficulty, lay in obtain-
ing fresh pig stomach) and further work with this medium might have produced better results. This
medium is a modification of Martin's broth, the first medium incidentally in which growth of the
bovine pleuropneumonia organism was observed. Edwards (unpublished work) working with a
strain of foreign origin, used a similar medium in his studies on the bovine pleuropneumonia organism
at Mukteswar and claimed excellent results. Purchase (personal communication) has evolved a
special cooked blood-agar medium (formula not stated), which is easy to prepare and on which colonies
of the bovine pleuropneumonia organism develops satisfactorily. We have found that minute colonies
just visible with a pocket lens (X 10) develop in ordinary cooked blood-agar slants, provided the
medium is freshly made and such medium sown from a growth well established in Bennett's serum
broth. However, this medium could not be recommended for routine work. Conditions of reduced
oxygen tension (+10 or +20 per cent) do not enhance the value of Bennett's serum agar medium,
SUSCEPTIBILITY
Bovines
(a) Kumaoni cattle. These are highly susceptible to the subcutaneous inoculation of crude
lymph virus. The percentage of 'Takes' is considerably higher than that recorded in Kenya cattle
[Purchase, 1939]. Although the Kumaon Hills are known to be free from contagious bovine pleuro-
pneumonia, yet the reaction of local animals to subcutaneous inoculation of the Assam 1 virus (crude
lymph) is somewhat erratic. Thus of a total of fifty-nine cattle used in the propagation of the
virus, the results in fifty-three animals that reacted and died were:
Incubation period
5 days and under
.
.
.
.
.
.
.
6-10 days
.
.
.
.
.
.
.
.
.
11-15 „ . . . . . . . .
16-30 „
.
.
.
.
.
.
.
.
No. reacting
31
15
6
1
Reaction period
6-14 days
10-34 „
22-30 „
49
Average
9.3 days
17.6 „
26.6 „
....
The reaction period is the interval between the first sign of local reaction at the site of inoculation
and death or destruction in the moribund state. The temperature reaction commenced, on an average
two days after the first appearance of the local reaction. It is evident that Kumaoni cattle are
highly susceptible to the virus, even if the type of reaction tends to be erratic. In the present test
the susceptibility ration is 93.2 per cent with 90 per cent mortality in all animals inoculated. The
nature of the reaction did not vary with the amount of inoculum, one drop of the virus was as effective
as several hundred times this amount. The period of storage of the virus, which was always under