Loss of pathogenic potential after cloning of the low-passage Borrelia burgdorferi ZS7 tick isolate: a cautionary note

To study clonal polymorphism of Borrielia burgdorferi antigens in the cours e of an experimental infection sequence, the low-passage tick isolate ZS7 w as cloned by two rounds of agar subsurface plating. The resulting clones sh owed a variable pathogenic potential after experimental infection of C.B-17 .scid mice. The test clone 4.2.II: selected for virulence by two passages i n immunodeficient scid mice, failed to establish a successful infection in immunocompetent AKR/N mice, indicating the loss of pathogenicity traits req uired for evasion of the specific immune response. Cloning of natural or cl inical B. burgdorferi isolates is a prerequisite for analyzing genetic and antigenic variation of the pathogen. However, the inevitable propagation in artificial media during cloning may lead to a loss of pathogenic features rendering the subsequent experimental infection of animals impossible. A co mbined procedure of in vitro cloning and in vivo selection also does not so lve the dilemma because B. burgdorferi variants arise by recombinatorial pr ocesses in the pathogen's dynamic genome during the course of infection. Co nsequently, the resulting bacterial isolates from infected animal tissues r epresent again non-clonal, heterogeneous B. burgdorferi populations. In pri nciple, cloning of a B. burgdorferi population is the appropriate method to analyze the polymorphism of individual molecules during infection. As a ca veat, however, one has to envisage that during propagation of individual cl ones in vitro and in vivo independent genetic variations (epigenetic or mut ational) may occur with consequences on the virulence of the clones. This m ay complicate the delineation of a clear correlation between the antigenic polymorphism observed and the change of virulence.