Norris S J, Howell J K, Garza S A, Ferdows M S, Barbour A G
Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston 77225, USA.
Infect Immun. 1995 Jun;63(6):2206-12. doi: 10.1128/iai.63.6.2206-2212.1995.
Borrelias that cause Lyme disease lose the ability to infect and cause disease in laboratory animals following 10 to 16 passages of in vitro culture. In this study, clonal populations of the Sh-2-82 (Sh2) and B31 strains of Borrelia burgdorferi were isolated by subsurface plating on BSK-II agar plates and examined for infectivity in the C3H/HeN mouse model. Mice were injected intradermally with 10(5) B. burgdorferi organisms, and the tibiotarsal joint, heart, and bladder were cultured 2 to 4 weeks postinfection to determine whether viable organisms were present. Clones exhibited either a high-infectivity phenotype, in which cultures were consistently positive at all organ sites, or a low-infectivity phenotype, in which a low proportion of cultures were positive (5 of 40 in a representative experiment). In an Sh2 population that had undergone five in vitro passages, 7 of 10 clones were of the high-infectivity phenotype, and the remaining clones were of the low-infectivity phenotype. The proportion of high-infectivity clones decreased with continued in vitro passage, with only 1 of 10 clones exhibiting the high-infectivity phenotype after 10 passages and 0 of 10 clones yielding positive cultures after 20 passages. Representative high- and low-infectivity clones from passage 5 Sh2 cultures had 50% infectious doses of 1.8 x 10(2) and 1 x 10(5), respectively. Subclones consistently reflected the same infectivity phenotypes as those of the parent clones. The protein profiles and plasmid contents of the high- and low-infectivity clones were compared and exhibited few discernible differences. On the basis of these results, the loss of infectivity during in vitro culture results from the outgrowth of low-infectivity clones and begins to occur within the first five in vitro passages. Further examination of clonal populations may lead to the identification of genetic and protein factors important in the virulence and pathogenicity of Lyme disease borrelias.
导致莱姆病的疏螺旋体在体外培养10至16代后，失去感染实验动物并引发疾病的能力。在本研究中，通过在BSK-II琼脂平板上进行亚表面铺板，分离出伯氏疏螺旋体Sh-2-82（Sh2）和B31菌株的克隆群体，并在C3H/HeN小鼠模型中检测其感染性。给小鼠皮内注射10⁵个伯氏疏螺旋体，在感染后2至4周培养胫跗关节、心脏和膀胱，以确定是否存在活的病原体。克隆表现出高感染性表型（所有器官部位培养物始终呈阳性）或低感染性表型（低比例培养物呈阳性，在一个代表性实验中为40个中的5个）。在经过5次体外传代的Sh2群体中，10个克隆中有7个具有高感染性表型，其余克隆具有低感染性表型。随着体外传代次数的增加，高感染性克隆的比例下降，传代10次后10个克隆中只有1个表现出高感染性表型，传代20次后10个克隆中没有一个产生阳性培养物。来自第5代Sh2培养物的代表性高感染性和低感染性克隆的半数感染剂量分别为1.8×10²和1×10⁵。亚克隆始终反映出与亲本克隆相同的感染性表型。比较了高感染性和低感染性克隆的蛋白质谱和质粒含量，几乎没有明显差异。基于这些结果，体外培养过程中感染性的丧失是由于低感染性克隆的生长，并且在最初5次体外传代内就开始出现。对克隆群体的进一步研究可能会导致鉴定出对莱姆病疏螺旋体的毒力和致病性重要的遗传和蛋白质因子。
