STUDIES ON THE CULTIVATION OF EQUINE INFECTIOUS ANEMIA VIRUS IN VITRO

I. SERIAL CULTIVATION OF THE VIRUS IN THE CULTURES OF VARIOUS HORSE TISSUES

Abstract

It has been known that equine infectious anemia caused by a virus. The experimental study of this disease has been severely handicapped by the lack of suitable experimental animals susceptible to the virus. It would facilitate the study of the disease enormously, if the virus could be cultivated in vitro. This has not been possible so far.
The experimental study of the virus cultivation through tissue culture method was carried out by the author since 1955.
The primary results are described in the present paper. Eight experiments of virus passage were performed respectively using various horse tissue with three culture methods. The plasma clotting roller tube culture (RT), the trypsin-dispersed monolayer cell culture (ML) and Maitlandtype tissue culture (MT) were employed.
The mixture of 30% horse serum, 10% CEE and 60% Hanks' BSS or 30% bovine serum and 70% synthetic medium No. 199 were used mainly as nutrient media. Infected horse sera (Goshun and Wyoming strain) collected at the acute phase were used as the original virus material. The material was inoculated into the culture in which excellent outgrowth was shown (RT and ML), or into the culture immediately after the cultivation (MT).
The serial cultivation and horse inoculation test were performed using the cultured materials. Cytopathic changes of the inoculated culture were observed microscopically under low magnification (RT and ML). The results are as follows.
1) Outgrowth of 16 kinds of tissues in RT culture exhibits different growth rate and cell type. Spleen, lymph node, Testis, and others showed active growth of fibroblast-like cells, while epithelial cell growth was manifested in kidney, lung and liver.
2) In four serialcultivations (RT, Exp. 1-4) using several series of spleen, lymph node, kidney, lung and adrenal gland, 4 or 5 and 7 day intervals of virus passages were carried out until 3-8 generations. In a series of kidney ML culture (Exp. 5), the virus passage was also performed until 4th generation adopting 4-5 days' interval. No cytopathic changes were observed through the experiments. The results of horse inoculation tests of the cultured materials produced positive infection only first-third generation. From the results, it may be presumed that total dilution of test material induced positive infection did not over the infective titer of original inoculum. Therefore, it was considered that the virus growth was negative in those culture.
3) In the serial passage of Exp. 6 (MT) including the series of spleen, lymph node and adrenal gland, 10 days' interval was adopted, and 2nd generation of the cultured material resulted positive infection by the horse test, but advanced passage materials showed negative. However, in two series of the virus passage using liver MT culture which were adopted 14 day (Exp. 7) and 7 days' interval (Exp. 8), the 4th (Exp. 7) and 8th (Exp. 8) generation of cultured materials caused positive infection respectively. According to the results, in the liver MT culture, it was suggested that the growth of the virus was rather positively in the relation of original virus infective titer.
However, the cultured material in advanced passage resulted negative, and furthermore, 8th generation material which induced a positive infection in primary test resulted negative by the retest at 6 months later.
The results were considered that the virus in the cultured material was inactivated during the preservation under unsuitable condition especially in a high level of pH.
4) Three samples of virus sera (Wyo strain) including the one used in the series of experiments (Exp. 4, 6, 7 and 8) resulted positive infection respectively at 10^-5 dilution, but was negative at 10^-7 by the horse inoculation tests.