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Bovine on Pleuropueumonia
was also observed in cultures of the Australian 264 organism. The morphology of the organism
has been studied in Berkefeld V filtrates incubated 12-48 hours. Overnight Giemsa has proved the
most satisfactory stain. Our observations on the morphology of the Assam organism correspond
so closely with standard descriptions [Ledingham, 1933; Tang et al, 1935; and others] that only
the following essential features need be given. There are four main growth phases : 1. Extremely
delicate filaments are just visible at a magnification X 1,000. Within the filaments and especially
at the terminations, tiny nodes are seen. The filaments undergo dichotomous branching and
ripening of the nodes to spores is characteristic of this stage. A fine mucoid sheath, staining bright
pink, is condensed at the centre of the branching process and invests the filaments for a short distance
(Plate IX fig. 5).3. The filaments disintegrate, releasing masses of ripe spores. These are often held
together by the mucoid substance to form small streptococcal-like chains. 4. Maturation is succeeded
by germination of the spores. These swell and acquire triangular, circular and somewhat polyhedral
shapes. On the contour, especially at angles, new chromatin condensations appear. From these
germinal centres new filaments develop. The 'asterococcal' forms of the organism, e.g. filaments
radiating from a germinal centre, may be fairly easily seen in Giemsa. stained films prepared from
the sediment of fresh lymph virus centrifuged at a speed of not more than 3,000 revolutions per minute
for 15 minutes.
(C). Viability of the organism: tenacity
1. In lymph virus. Fresh lymph virus stored at +5°C. and at room temperature (13º—24ºC.)
was tested, by the subcutaneous route, in cattle at 2-3 week's interval. The material stored at +5°C.
proved virulent at 3 months, but avirulent at 3 months and 25 days. The animal tested at this
interval failed to react and proved solidly immune to a later immunity test in which the control
reacted fatally. The material stored at 13°—24°C. killed at 18 but not at 35, 52, 69 and 92 days
although some reaction was noted in animals surviving the inoculation. The survivors were put
to an immunity test in which animals inoculated with 35,52 and 69 day virus proved solidly immune
against fatal reactions in 3 controls. The animal used in the 92 day test succumbed, but it is to be
noted that in this case an interval of 1 month and 2 days elapsed between inoculation and immunity
test, whereas this interval was 2-3 months in the other animals of the batch.
2. In culture. Cultures of the Assam organism retain their viability for long periods when
stored at room temperature (13-24°C. Mukteswar), at incubator temperature (37.5°C.) and at +5°C.
Four tubes of Bennett's serum broth were inoculated with a Berkefeld V filtrate of fresh lymph virus
and incubated for five days at 37.5°C. when growth was fully developed. One lot of eight tubes
was stored at 5°C. a second at 37.5°C. and a third at room temperature. Subcultures from each
lot were made at intervals of 14 days in Bennett's broth, one slant agar and one ordinary broth tube
being simultaneously inoculated as a control. The original cultures under all these conditions of
storage maintained their viability for not less than 188 days; this period was probably short of the
actual, but on account of accidental contamination of the cultures, observations could not be extended.
Two cattle were subcutaneously inoculated with 4 c.c. 120-day old culture stored at 5ºC. One animal
reacted typically and died and the second failed to react. The organism in Bennett's broth is ther-
molabile; it can survive one hour (maximum period tested) at 45°C. 10 minutes but not 20 at 50°C.
The organism dies if held for 10 minutes at 55°C.
IMMUNITY
Formalinised lymph virus vaccine. The vaccine was prepared as follows : 20 c.c. fresh, virulent
lymph virus was diluted with 10 c.c. M/15 phosphate. The coarse particles were removed by straining
through muslin, and 0.3 c.c. of a 10 per cent formalin in distilled water was added to give a final
concentration of 1/1,000. The sample of formalin used was assayed to contain 40 per cent formal-
dehyde. The pH of the experimental vaccine was finally adjusted at 7.4. The vaccine was divided
in two batches, one being stored for two days and the other for six days at 25°C. Cultural test of
the two-day vaccine showed that the concentration of formalin used had destroyed the organism.
Four cattle were tested with the vaccine; two were subcutaneously inoculated at the point of the
shoulder with 6 c.c. two-day vaccine and two similarly inoculated with the same dose of six-day