Abstract
In the previous paper the author reported that the propagation of equine infectious anemia virus was noticed in trypsinized bone marrow cell culture showing the cytopathic changes of the round cells. The present paper describes that the virus propagation was also comfirmed in the cultures of peripheral blood leucocytes of horse. The leucocyte culture was performed by following method. The heparinized blood was collected from jugular vein of healthy young horses, and allowed to stand at room temperature for 15-20 minutes, then the plasma was removed. Leucocytes were collected from the plasma by low speed centrifugation, and washed once in cold Hanks' solution. Then, the leucocyte sediment was resuspended into the nutrient medium in concentration of approximately 107per ml, and cultivated stationally at 37°C. Modified carrel flask was used containing each 2ml of the cell suspension. Mixture No. 199 with 40% bovine serum was used as nutrient medium.
Infected horse sera (Wyo. strain) collected during the acute phase were used as original virus materials, and healthy control sera were obtained from the same horses at immediately before the virus inoculation and the both serum materials were used in parallel with each other.
Virus inoculation, observation of the culture and serial cultivation were performed by mean of bone marrow culture method described in the previous report.
Results obtained are as follows.
1) Horse leucocytes were cultivated on glass wall during 2-3 weeks, but the cell multiplication was not recognized.
2) On 7-9 days after the virus inoculation, the cultured cells manifested the similar cytopathic changes to the ones which ascertained on the round cells of horse bone marrow culture.
3) The fluids harvested from virus inoculated cultures which appeared the C. P. changes reproduced the similar changes in the following cultures by transfer inoculation. Consequently, serial cultivation of the virus was carried out through 154 days in 17 generations by checking sign of the C. P. changes. In despite of the lack of C. P. changes in control culture inoculated with the healthy horse serum, the culture fluids were transfered successively in parallel with that of virus passage series.
4) Horse inoculation tests were carried out using the serially cultivated materials, and results obtained are as follows. Testing each 1ml of the materials of 7th and 14th generation and 1ml of 10-1 diluted material of 17th generation in the virus passage series the infectivity against horse were demonstrated as positive, but, 1ml of the material of 14th and 17th generation in the control passage series did not induced any sign of disorder in inoculated horses. The dilution of the last inoculated material in the virus series was calculated 10-23 of original virus material by serial passages except the medium exchange in total 70 times performed during the successive serial cultivations.
As the results described above, it may be concluded that equine infectious anemia virus propagated successively in horse leucocyte culture and appearance of cytopathic changes would be accompanied with the virus propagation.
