Abstract
In order to cultivate in vitro equine infectious anaenaia (ERA) virus with equineleucocyte culture, it is absolutely indispensable to gain a prosperous cultrue of equineleucocytes themselves. The following experiments were performed to attain these purposes.Heparinized blood samples were collected from 13 healthy horses 1 -2 years old andemployed to remove blood plasma. Leucocytes harvested by centrifugation of the plasmawere suspended in a culture medium cornposed of media No. 199 plus 40% bovine serumand incubated at 37C. The results of leucocyte cultivation were compared among thehorses from which the cells had been collected (Tables l and 2).The results were strikingly diverse among the individuals, indicating that the culturalharvest seerned quite dependent upon individuals. In particular, more or less sufficientgrowth of leucocytes was found in only a few horses. This finding seemed to be some-what similar to the discription of Kobayashi et al.However, the interval between the initiation of growth and the cornplete fall ofdegenerated cells was [1 days at the longest. This interval of time was fairly short, as compared with that reported by Kobayashi et al., or 21 days.Such poorly grown and short-living cultures of leucocytes were ernployed to cultivateETA virus at intervals of 5 -7 days. Reversion tests were also carried out on horseswith the cultured virus. In these tests, cultivation in vitro of both strains of ETA virus, Wyoming and Goshun, was proved to be positive until the 6th passage. Judging fromthis result, the cultivated equine leucocytes were considered to have a fairly sharpsusceptibility to the virus strains.The infective titre of the virus was determined with the original and diluted (at10, 10, and TO ) suspensions of the 5th-passage culture of the Wyoming strain. As a result, only the original suspension was positive in action to the horse, which manifestedstrikingly mild clinical signs. These findings seem to indicate that the proliferation ofETA virus cultivated in poorly grown leucocyte culture is carried out at only a remark-ably low level.Furthermore, alternative passages of ERA virus were tried between tissue cultureoriginated from an equine embryo and equine leucocyte culture, but in vain. Theseresults were considered to be due to the use of poor growth of cultured leucocytes.a result, only the original suspension was positive in action to the horse, which manifestedstrikingly mild clinical signs. These findings seem to indicate that the proliferation ofETA virus cultivated in poorly grown leucocyte culture is carried out at only a remark-ably low level.Furthermore, alternative passages of ERA virus were tried between tissue cultureoriginated from an equine embryo and equine leucocyte culture, but in vain. Theseresults were considered to be due to the use of poor growth of cultured leucocytes.
