50
Vaccination against Ranikhet Disease
It will be observed from Table V that during the first 50 passages the virulence
was partially lost ; the change being more or less comparable to that obtained by Iyer
with his line II (our R2). Further passages produced no marked loss of virulence, and
what originally appeared to be a hopeful line of advance was abandoned after over 300
passages.
The net result of the work done under this heading was that eggs were incubated
after inoculation for 48 hours as a routine and the virus then collected.
Incubation of eggs at 41.5°C.
Incubating the eggs at 41.5°C. was tried but at the 15th passage the virus was
still fully virulent.
Heating of the virus
The effect of heating the virus was examined. The virus to a final dilution of 1:10
and 1:50 was added to water raised to the temperature required. From these experiments
no results of practical importance emerged, but it was noted that at all temperatures
tried the virus remained alive much longer than previously reported. Thus, at 55°C. the
virus was still viable after three hours, at 60°C. after one hour, and at 70°C. after 15
minutes. At 60°C. the virus after 1¼ to 1¾ hours' heating appeared to undergo a change,
When inoculated into eggs the embryos died in three to seven days with typical lesions
of Ranikhet disease and usually also with marked skin oedema. There were also a few
faint follicular lesions. Sub-inoculation into fowls either from first or second passage
from such embryos produced no reaction or immunity. That the virus was not dead
was shown by the fact that direct inoculation of the heated material into chickens and
fowls gave results varying from acute affection, solid immunity without reaction, to no
reaction and no subsequent immunity.
Gel virus as seed material
It appeared from the results of the gel vaccines that it might be possible to re-
cover virus in an altered form at some stage in its production. Several attempts were made
and the results often conformed with those obtained during the heated virus experiments
just mentioned, i. e. embryos were obtained showing typical lesions from which sub-inocula-
tions in eggs were negative or only weakly positive. In no case could the infection be trans¬
ferred from these embryos to adult fowls.
In one instance a strain comparable to those found in the heated virus and gel
virus experiments was isolated from drinking water from an infected pen. No exhaus-
tive investigation was made, but the occurrence of such strains may perhaps be a factor
in the preservation of the few birds which survive during a natural outbreak.
Mixture of virus and serum, etc.
Small-scale experiments were carried out at incubator temperatures with mixtures of
virus and antiserum, virus and bile, virus and crystal violet, and virus and glycerol. With
the first three, the indication was that viruses were produced which behaved somewhat
like those of the gel and heated viruses. When mixtures of virus and glycerol were
inoculated on to embryos, virus could not be re-isolated.
Testing of field strains by egg passage
Iyer (loc. cit.) has suggested that strains from different outbreaks might vary in be-
haviour during egg passage. To examine this point, eight strains were passaged at about
monthly intervals, whenever eggs were available. After about 30 passages there was
no change. The matter, however, is not regular. Thus with strain R1, which on account
of its high virulence has been maintained as our stock strain in fowls, it was found
in one instance that some loss of virulence had occurred from as few as three egg
passages. In another trial, the same strain was passaged 54 times without visible
change. For this, no satisfactory explanation can be offered. The possibility that atte-
nuation may have been due to contaminating bacteria or fungi was examined with en-
tirely negative results. In one case it appeared that eggs from freshly vaccinated fowls
might have been a factor.
In addition to the strains mentioned above, we maintained three sub-lines of Iyer's
line II, designated R2B, R2C and R2D. Iyer (in the press) has reported the results of a
fairly large test of the 92nd egg passage of R2B, in which the casualty rate was about
45 per cent or over. These strains were again tested by us at the 114th, 103rd and
87th passages respectively, using two fowls each time. In the case of R2B, both fowls
reacted slightly and recovered, with R2C the reaction was more severe and one died, and