Isolation of moderately infectious Borrelia burgdorferi sensu stricto from attenuated cultures by using complement-mediated, antibody-dependent lysis selection technique in a mammalian tissue co-culture system.
We investigated the association between complement resistance and phenotypes of pathogenicity of Borrelia burgdorferi sensu lato isolates cultivated in a LEW/N rat tibiotarsal joint-derived tissue feeder layer-supported co-culture system. Guinea pig complement and immune serum raised in LHS/Ss hamsters caused complete lysis of B. burgdorferi sensu stricto isolate 297, B. afzelii and B. garinii in Barbour-Stoenner-Kelly's medium; however, tissue co-cultured B. burgdorferi sensu stricto contained complement escape variants. The arthritogenicity and infectivity of these variants were tested in 3-week-old Syrian hamsters and in a vaccinated hamster model in which formalin-killed B. burgdorferi sensu stricto C-1-11 vaccinated animals develop severe arthritis after challenge with live, pathogenic, low-passage 297 isolate. Non-animal-passaged complement escape variants were infectious in both animal models as demonstrated by re-isolation from the infected animals and competitive PCR. IP injection of animal-passaged complement escape variants caused development of severe arthritis in vaccinated animals 5 weeks post-injection; animal passage of complement escape variants was necessary for isolation of arthritogenic spirochetes from high-passaged, non-arthritogenic, attenuated borrelia cultures. Complement escape variants synthesized outer surface protein E as demonstrated by SDS-PAGE and western blotting analyses. The complement-mediated selection technique in tissue co-culture provides a novel approach to the studies of Lyme disease, enables us to isolate pathogenically distinct borrelia populations from attenuated cultures and prepare a moderately infectious, non-pathogenic live vaccine against this illness.
Sen
Ece
E
Department of Microbiology, Medical School, Yeditepe University, Kayisdagi Campus, Kayisdagi cad. Erenkoy 34755 Istanbul, Turkey. ecesen@bluemail.ch
Schell
Ronald F
RF
D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology*; D000942:Antigens, Bacterial / Q000096:biosynthesis; D001170:Arthritis, Infectious / Q000276:immunology / Q000382:microbiology; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis; D001426:Bacterial Proteins / Q000032:analysis / Q000096:biosynthesis; D001433:Bacteriolysis; D015153:Blotting, Western; D025065:Borrelia burgdorferi / Q000276:immunology / Q000302:isolation & purification* / Q000472:pathogenicity*; D015748:Borrelia burgdorferi Group / Q000276:immunology; D018929:Cell Culture Techniques; D018920:Coculture Techniques; D003165:Complement System Proteins / Q000276:immunology*; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D007106:Immune Sera; D008074:Lipoproteins / Q000096:biosynthesis; D008193:Lyme Disease / Q000276:immunology / Q000382:microbiology; D022123:Lyme Disease Vaccines / Q000276:immunology; D051381:Rats; D014611:Vaccination; D014774:Virulence
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D007106:Immune Sera; D008074:Lipoproteins; D022123:Lyme Disease Vaccines; C084998:OspE protein, Borrelia burgdorferi; D003165:Complement System Proteins