16
Studies on Newcastle (Ranikhet) Disease Virus
Experimental procedure
Vigorous, healthy fowls were specially mated for this work. Leghorns
and Rhode Island Reds were used. Fertile eggs, not more than five days old,
were selected for incubation. From 6 to 12 eggs at a time were placed in the
incubator and three such sets were made in a week, so that sufficient eggs
might be available each time that inoculation was due. After incubation for
9 to 12 days, the eggs were tested by ' candling ' to remove ' clears ' and
' dead-in-shells '. The optimal period of incubation for an easy technique of
inoculation and collection of virus was found to be about 11 days.
The technique of Burnet [1933 and 1936] was followed as far as possible'
Experimental infection of eggs
Virus. The first batch of eggs was inoculated with a Berkefeld V filtrate
of liver and spleen fowl-passage virus which has undergone, during seven years,
innumerable and continuous, as well as intermittent passages, through fowls.
The inoculum was tested for bacterial contamination by appropriate cultural
tests. Usually three eggs were inoculated with the material at each passage.
The records were maintained by noting the passage number as P1, P2, etc.
and the number of eggs within each passage as E1, E2, etc. The virus was
found lethal to the embryo at the first passage and also during the subse-
quent transfers, using every time-for sub-inoculation, saline or broth emulsion
of the infected chorio-allantoic membrane or the whole embryo. As a rule,
direct sub-inoculations were made with fresh virus each time but occasionally,
when suitable well-developed eggs were not available for the work, egg virus
in the form of emulsion of chorio-allantoic membrane or embryo in broth
or saline was stored in the refrigerator (0 to 5°C.) until satisfactory eggs became
available. Storage of virus for one or two weeks did not appear to alter its
virulence for the embryo. No difficulty was experienced in obtaining quan-
tities of virus, free from bacterial contamination, at each passage. It was
also observed that contaminated samples could be purified by filtration through
a Berkefeld V filter without any appreciable loss of virulence. The age of the
embryo at inoculation ranged from 9 to 13 days. Eggs were opened for ex-
amination and collection of virus on the second or third day following rein-
cubation after inoculation. It should be mentioned that a re-incubation of
the egg of over three days after inoculation often resulted in partial or com-
plete loss of virulence of the virus. Therefore, the period necessary for the
reincubation of eggs after inoculation with virus seems an important factor
for a successful propagation of the virus [Brandly, 1937].
Following inoculation and re-incubation, the eggs were, as a rule, opened
for examination and collection of the virus on the 2nd or 3rd day. Death of the
embryo usually occurs within 48 hours. In the case of dead embryos,
the shell membrane which can be seen through the cover slip is usually moist
and greyish. There is, as a rule, transudation of fluid between the shell
membrane and the chorio-allantois. The latter is thickened and oedematous
and often shows areas of cloudiness resulting from cellular infiltration (Plate IV,
figs. 2 and 3). The membrane also shows minute greyish white flecks
which Burnet described as the areas of ectodermal proliferation characteristic
of Newcastle disease, Histological sections of the chorio-allantois, as described