12
Studies on Newcastle (Ranikhet) Disease Virus
(iii) Formalized spleen vaccine. That formalin is invaluable in the prepa-
ration of vaccines for virus diseases has long been known, consequently Shirlaw
[1937] suggested the use of formalized spleen vaccine for Ranikhet disease as
in the control of yellow fever. Bedson [1938] reported that a considerable
degree of immunity to psittacosis, in mice could be produced by the injection
of a vaccine consisting of virulent mouse spleen emulsion inactivated by 0.1
per cent formalin and stored in the refrigerator for 10 to 14 days before use.
Preparation. The entire spleen usually about the size of a cherry and
weighing about four grams, was collected from a fowl which had died of experi-
mental infection with Newcastle virus. A 5 per cent suspension in phosphate
buffer solution pH 7.6 was prepared and 0.1 per cent formalin added. The
contents were thoroughly mixed and the vaccine-mixture was stored in the
refrigerator at 0 to 5°C. for 14 days. It should be mentioned that, while prepar-
ing the vaccine, the whole spleen was included without filtration or centrifu-
gation to get rid of the coarse tissue fragments but the tissue was finely minced.
This was done as it was thought that virus, as contained within the cells and
used with the tissues, would offer a more efficient product than the usual cell-
free organ extracts. Kelser [1930], referring to the rabies-chloroform-treated¬
vaccine, considered that the active principle of the vaccine may be something
other than dead virus, possibly a cellular principle resulting from the reaction
between the virus and the tissue cells or it may be a particular stage of the
virus which occurs only in close conjunction with the tissue cells.
Test. The formalized spleen vaccine for Newcastle disease was injected
in fowl No. 2089, 5 c.c. subcutaneously on 4 April 1940 ; the bird survived and
on 18 April 1940 it was exposed to coop-contact infection. The fowl died
on 23 April 1940 showing lesions of Newcastle disease.
(b) Crystal violet vaccine
The use of crystal violet by Dorset [1935] in the United States Bureau of
Animal Industry, as an agent for the attenuation of hog cholera virus, yielded
promising results for the production of a satisfactory vaccine. The product, de-
signated as regular vaccine Mc Bryde & Cole [1936] was prepared by adding
one part of one per cent aqueous solution of phenol to nine parts of denfirinated
blood obtained from pigs sick with cholera. Five parts of one per cent aqueous
solution of crystal violet were then added to 100 parts of the previously pheno-
lized blood. This mixture was incubated at 37.5°C. for 14 days.
Crystal violet vaccine for the prevention of swine fever in pigs has given
variable results in the hands of different workers, Mc Bryde & Cole [1936],
Munce [1937], Hupbauer and Tomasec [1937] and Edgington and
Schalk [1939], but certain improvements in the physical and chemical nature of
the vaccine appear to be necessary.
(i) Preparation of crystal violet vaccine for Newcastle disease virus.
Only fresh virus was used in the preparation of the vaccine. Simultaneously
with the study of the immunizing value of the vaccine, the effect of crystal
violet on the virus after different periods of contact at 37°C, and at room
temperature was determined with a view to developing a standard in the pre-
paration of the vaccine.
Crystal violet solution. Crystal violet of commercially high purity, from
Messrs G. T. Gurr & Co., London, was used. A 0.5 per cent aqueous solution