da Silva R, Sacks D L
Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892.
Infect Immun. 1987 Nov;55(11):2802-6. doi: 10.1128/iai.55.11.2802-2806.1987.
The in vivo virulence patterns of promastigote populations defined on the basis of agglutination by the lectin peanut agglutinin (PNA) were studied for various cloned lines of Leishmania major. Promastigotes derived from logarithmic-phase cultures, which were routinely 100% agglutinated at 100 micrograms of PNA per ml, were relatively avirulent for BALB/c mice. The relative virulence of stationary-phase promastigotes appeared to be attributable to the proportion of nonagglutinable (PNA-) promastigotes contained within these populations. Purification of PNA- organisms from stationary cultures provided for each clone the most virulent inoculum, supporting the view that this change in lectin binding accurately reflects the development of infective metacyclic stage promastigotes. By studying this marker, we found that there was considerable variation in the degree to which different strains and clones underwent metacyclogenesis during growth. Examination of a reportedly avirulent L. major clone revealed that metacyclogenesis was unusually delayed and inefficient for this clone, but that those PNA- promastigotes which could be recovered from late-stationary-phase cultures were virulent for BALB/c mice. The loss of virulence associated with frequent subculture could also be attributed to a drastic diminution in metacyclogenesis potential over time. A clone which yielded over 90% PNA- promastigotes during growth within passage 1 generated fewer than 10% PNA- promastigotes during growth by passage 94. Subcloning of late-passage attenuated promastigotes yielded a clone for which no PNA- promastigotes could be generated during growth, and an infective population could not be derived from this clone. Thus, metacyclogenesis does not appear to be stable for even cloned lines of Leishmania promastigotes, and virulence comparisons between different strains and clones can be meaningfully made only if the metacyclic populations contained within the respective inocula are determined.
针对利什曼原虫的不同克隆系，研究了基于凝集素花生凝集素（PNA）凝集作用所定义的前鞭毛体群体的体内毒力模式。对数期培养物衍生的前鞭毛体，在每毫升含100微克PNA时通常100%凝集，对BALB/c小鼠相对无毒力。稳定期前鞭毛体的相对毒力似乎归因于这些群体中不可凝集（PNA-）前鞭毛体的比例。从稳定期培养物中纯化PNA-生物体，为每个克隆提供了最具毒力的接种物，支持了凝集素结合的这种变化准确反映感染性后循环期前鞭毛体发育的观点。通过研究这个标志物，我们发现不同菌株和克隆在生长过程中经历后循环发育的程度存在相当大的差异。对一个据报道无毒力的利什曼原虫克隆进行检查发现，该克隆的后循环发育异常延迟且效率低下，但从稳定期末期培养物中回收的那些PNA-前鞭毛体对BALB/c小鼠具有毒力。与频繁传代相关的毒力丧失也可归因于后循环发育潜力随时间的急剧降低。在第1代传代生长期间产生超过90% PNA-前鞭毛体的一个克隆，在第94代传代生长期间产生的PNA-前鞭毛体少于10%。对传代后期减毒的前鞭毛体进行亚克隆，得到一个在生长过程中不能产生PNA-前鞭毛体的克隆，并且不能从该克隆获得感染性群体。因此，即使是利什曼原虫前鞭毛体的克隆系，后循环发育似乎也不稳定，只有在确定各自接种物中所含的后循环群体时，才能对不同菌株和克隆之间的毒力进行有意义的比较。
