新生隐球菌格鲁比变种临床株与环境株微卫星基因型的小鼠毒力实验研究

Virulence analysis between environmental and clinical isolates of Cryptococcus neoformans var. grubii based on microsatellite patterns

目的了解巴西新生隐球菌格鲁比变种（C.neoformans var．grubii）多位点微卫星定型（ multilocus microsatllite typing, MLMT）在临床和环境中的分布特点，探讨不同微卫星型的新生隐球菌格鲁比变种临床株和环境株的毒力差异。方法根据多位点微卫星分型技术对分离自巴西的40株（17株临床株和23株环境株）新生隐球菌格鲁比变种进行分型，筛选出临床株及环境株中分布占优势的菌株，应用小鼠毒力试验分别对其进行毒力检测，通过发病情况及病理切片比较毒力差异。结果40株格鲁比变种共鉴定出11种微卫星型，临床株中的优势菌株为MLMT-13型，共9株，占临床株的52．9％（9／17），环境株中的优势菌株为MLMT－36型，共10株，占环境株的43．5％（10／23）；小鼠毒力试验结果显示MLMT－13型感染小鼠后发病率为100％，而MLMT－36型发病率为7．5％，且病理结果也存在明显差异。结论格鲁比变种在从环境迁徙到宿主的过程中，毒力随环境的变化发生了改变，该变化也可在其微卫星重复序列上体现出来。该实验证明新生隐球菌格鲁比变种的分布及来源与微卫星型之间存在相关性，临床株MLMT-13型和环境株MLMT－36型之间存在着显著毒力差异。

Objective To investigate the genetic relation between Cryptococcus neoforrnans var. grubii clinical and environmental isolates in Sao Paulo, Brazil, and to test the virulence difference between theclinical strains in MLMT - 1 3 genotype and the environmental strains in MLMT - 3 6 genotype . Methods Multilocus microsatellite typing （MLMT） method was applied for the genotype analysis in our study. Through this method, we recognized two genotypes that distinguish a majority of clinical and environmental strains. In order to compare virulence between the two types, we chose to infect BALB/c mice （6 weeks, female） with 9 MLMT-13 strains and 10 MLMT-36 strains intravenously. Results Forty（ 17 clinical and 23 environmental isolates） were analyzed. Of 17 clinical strains, 9 belonged to a major type of MLMT-13 （52.9%）. They were mainly isolated from clinical specimens. About 43.5% of strains from the environ- ment belong to a major type of MLMT-36, which are indigenous to environments and which were not isolated from clinical samples. The mortality rate and pathological changes of the above mice were observed during two months after injection. The results showed that the mortality rate of mice infected with MLMT-13 strains was 100%, while the mortality rate with MLMT-36 strains was 7.5%. The pathological sections showed that lesions of MLMT-13 infected mice appeared in the brain, lungs, liver and kidneys, while the lesions of MLMT-36 infected mice only appeared in the brain. Most brains of MLMT-13 infected mice were distorted, and both the number and size of lesions in such brains were much larger than those of MLMT-36 infected mice. Conclusion Our study illustrated the virulent difference between MLMT-13 and MLMT-36, which are isolated from patients and environment respectively. The results inferred that some genetic changes, such as microsatellite repeats, might occur between environmental and clinical isolates through their environmental adaptation progress.