J. R. HADDOW AND J. A.
IDNANI
49
appeared to be fixed at a point where 40 to 50 per cent of birds inoculated either died
or were permanently injured, Various factors which may have led to this attenuation
in the egg are mentioned by Iyer and Dobson [1940], and, when circumstances neces-
sitated our taking over the work, we attempted to see how far these factors were ope¬
rative and if possible to define the process of attenuation more precisely. Unfortunately,
owing to war requirements our efforts had to be hastened and so were confined to lines
which seemed to promise quick results.
To begin with, it was necessary to organize a continuous supply of satisfactory
embryos and fully susceptible fowls, previous work at the Institute having been vitiated
by lack of these. Local difficulties in incubation and in transporting eggs from the Poultry
Research Section at Izatnagar 90 miles away also had to be overcome. Unless otherwise
stated, amniotic fluid was used in all cases for transmission work, since this has a fairly
constant virus content (m. 1. d. for fowls, 1 × 10 -6 ) and can be collected easily without
risk of contamination. It is convenient to use 0. 1 c. c. of the undiluted fluid as inocu-
lum in passage experiments, and this dose kills the embryo within 42 hours. Lower
dilutions kill embryos more slowly and at 1 × 10 -7 to 1 × 10 -9 death may be delayed
until the third or fourth day after inoculation. Deaths at longer intervals with virulent
virus have not been noted, but deaths after five to eight days have occurred with viruses
altered by artificial means and in these cases there is often a very noticeable oedema
of the embryo's skin.
Optimal interval for passage in eggs
Eggs were incubated for three, four and seven days after inoculating virus and
serial passages were made at those times. As these eggs invariably became contaminated
with moulds or bacteria, the three and four day series were stopped after the sixth and
fifth passages respectively, at which time there was no evidence of attenuation. The seven
day series was continued for 12 passages, by which time the virus had died out.
It was evident from intervening tests that the virus content of eggs decreased when
the period of incubation in each passage exceeded three days. In a parallel experiment
in which the amniotic fluid was kept in test-tubes in the incubator between each passage
the virus died out at the fifth passage.
The effect of passaging at short intervals has also been tested ; for this, 24 hours
was selected as suitable. The strain of virus, R 1, selected for this purpose, was one
which had already been passaged 65 times at two-day intervals without under-
going any change, [Iyer, 1941]. This strain was freshly isolated from fowls and then
passaged regularly at the shorter intervals. Table V gives the results.
TABLE V
Tests with 24-hours passaged egg virus
No passage
50
60
132
155
188
203
203
203
203
240
259
264
302
330
333
No. of fowls used
2
2
2
4
4
2 sub-inoculated from dead bird of
passage 188
5 (various dilutions)
3 (chicks)
2 sub-inoculated from dead chick
6
2 sub-inoculated from dead fowl
6
7 in contact with above
8 (various dilutions)
2 sub-inoculated with liver of dead
bird
3 (chicks)
4
2 sub-inoculated from destroyed
fowl
8 (various dilutions)
2
4 (chicks)
Results
Severe R ; recovered
D.
R ; recovered.
D.
R ; 1D.
R nil ; I
Slt R ; I.
Severe R ; 1D.
R nil ; I.
3 R ; 1D.
3 R nil ; I.
R nil ; I.
R nil; 51 (1D-before test).
2 R , 7 I.
3D ; 5 I.
1R ; both I.
2D.
4R ; 1 destroyed.
R ; I.
2D.
D.
D.
R = reacted; I = immune ; D = died.