STUDIES ON THE CULTIVATION OF EQUINE INFECTIOUS ANEMIA VIRUS IN VITRO

II. PROPAGATION OF THE VIRUS IN HORSE BONE MARROW CELL CULTURE

Abstract

In the previous paper, the author reported on unfavorable results of serial cultivations of equine infectious anemia virus using the cultures of various horse tissues in several methods. In this experiment, horse bone marrow culture was examined for the virus propagation. Bone marrow were collected mainly from femoral bone of healthy colts, and trypsinized. The resulting cells were suspended into the nutrient medium in concentration approximately 10⁷ per ml. Two ml of the suspension was placed into each modified carrel flasks and cultivated stationally at 37°C. Medium exchanges of the whole volume were carried out at every 1-3 days. For some experiments, the punctured bone marrow fluids were used, also, as a source of the cell culture. The mixture of 40% bovine serum and 60% synthetic medium No. 199 were employed as nutrient fluids in the majority of experiments, and the mixture of 30% horse serum, 10% CEE and 60% Hanks' solution were used in some cases. The infected horse sera (Wyo. strain) collected on the acute phase were used as original virus material, and healthy control sera were obtained from the same horses at immediatly before the virus inoculation. The both sera were inoculated 0.2ml into each cultures at 1 day after the cultivation, and serial passages were performed in parallel with each other. The cultures were observed microscopically under low magnification.
Results obtained are as follows.
1) Two kinds of cells in the bone marrow culture were morphologically observed. The one is round cells which was not ascertained its multiplication in vitro and cultivated on the glass surface for 2-4 weeks or more, while, the other is fibroblast-like cells which appeared 2-4 days after the cultivation and showed active cell growth. Cultures of punctured bone marrow fluids resulted poorly culture of round cells, but fibroblast-like cells were easily cultivated showing the active growth.
2) The cultures inoculated with virus sera manifested the cytopathic change after 1-2 weeks of the virus inoculation. The C. P. change was observed only in the round cells and no change was recognised in fibroblast-like cells.
3) The fluids which were harvested from cultures exhibited C. P. change reproduced the similar change in the following renewed cultures by transfer inoculation, and 8 passages through 81 days were performed by checking sign of the C. P. change.
4) Horse inoculation tests were performed using the cultured materials of 1st, 2nd, 3rd, 5th, 6th and 8th generation of virus passage series and of 1st, 5th, 6th and 8th generation of control passage series. Test horses inoculated with each materials of virus series infected positively in dosis of 1.0-0.001ml, but whole the ones inoculated with control series materials manifested no signs of infection injecting 1ml dosis of test material.
The dilution of the inoculated material of the last generation which indicated positive infection was calculated 10^-12 of original inoculum, even with the exception of 40 times exchange of medium in vessel, and was contrasted with the virus titer of the original inoculum, which was infective at 10^-5 dilution and not at 10^-7. From the above results it would be confirmed that the virus propagate in horse bone marrow cell culture.
5) The serial passage of the virus using the cultures of fibroblast-like cells from bone marrow fluids was advanced until 3rd generation adopting 14-16 days' interval, but the cultures showed no C. P. changes, and the horse inoculation test using the cultured material resulted positive in only the first generation and negative in further transfer.
As the results mentioned above, it may be considered that equine infectious anemia virus is able to propagate in the round cells with the indication of cytopathic changes, while, it seems to be negative in fibroblast-like cells which showed active growth in the culture.