Cultivation of viral agents from Crohn's disease. A new sensitive system

Abstract

The isolation and animal transmission of a viral agent from Crohn's disease patients stimulated studies aimed at the development of improved tissue culture techniques. The need for an effective tissue-culture system led to a comparison of African green monkey tissue-culture cells (A.G.M.K.), human diploid lung cells (WI-38), and a new tissue-culture line--continuous rabbit ileum (C.R.I.). Homogenates prepared from ileal specimens from four Crohn's disease patients and from four control patients without inflammatory bowel disease were filtered through a 0-2mu 'Millipore' filter. After confluence, groups of six tissue-culture flasks were inoculated with 0-3 ml of Crohn's disease or control filtrates. No cytopathic agents were isolated from A.G.M.K. tissue-culture. Cytopathic agents were isolated in WI-38 and C.R.I. tissue-cultures from each of the four Crohn's disease specimens but from none of the control specimens. A comparison of C.R.I. and WI-38 demonstrated that cytopathogenic change (C.P.E.) developed in C.R.I. earlier than in WI-38. C.P.E. was complete in a shorter period of time in C.R.I. but was irregular in WI-38. The sensitivity of WI-38 varied with the passage level and age of the monolayer. C.R.I. was found to be free of cytopathic adventitial agents upon inoculation of standard tissue-culture systems and weanling mice. Therefore C.R.I. is a sensitive and superior tissue-culture system for the cultivation of viral agents from Crohn's disease filtrates. The reproducible isolation of a viral agent from the ileum of patient's with Crohn's disease is confirmed by these studies.