John David E, Nwachuku Nena, Pepper Ian L, Gerba Charles P
College of Marine Science, University of South Florida, St Petersburg, FL 33701, USA.
J Microbiol Methods. 2003 Feb;52(2):183-96. doi: 10.1016/s0167-7012(02)00159-8.
Microsporidia are unique parasites recognized as a major cause of intestinal illness among immunocompromised patients and occasionally in otherwise healthy hosts. These organisms have been detected in water and are likely transmitted by the fecal-oral route. The most common human pathogenic microsporidia for which cell culture methods have been established is Encephalitozoon intestinalis. This study describes the development of a quantitative cell culture infectivity assay for E. intestinalis and its application to assess inactivation by ultraviolet (UV) light irradiation. The method described here employs calcofluor white, a fluorescent brightener that targets the chitin spore wall, to visualize groups of developing spores in order to confirm infectivity. Serial dilutions of the spore suspension were seeded into tissue culture well slides containing RK-13 cells. Slides were then rinsed, fixed in methanol and stained with calcofluor white and examined microscopically. Large masses of developing spores were easily visible on infected cell monolayers. Positive and negative wells at each dilution step were used to quantify the number of infectious spores in the original suspension using a most-probable-number (MPN) statistical analysis. This assay was used to evaluate the disinfecting potential of ultraviolet light on E. intestinalis spores in water. The ultraviolet dose required for a 3-log(10) or 99.9% reduction in the number of infective spores was determined to be 8.43 mW s/cm(2).
微孢子虫是一类独特的寄生虫，被认为是免疫功能低下患者肠道疾病的主要病因，在其他健康宿主中也偶有发现。这些微生物已在水中被检测到，很可能通过粪口途径传播。已建立细胞培养方法的最常见的人类致病性微孢子虫是肠脑炎微孢子虫。本研究描述了一种针对肠脑炎微孢子虫的定量细胞培养感染性测定方法的开发及其在评估紫外线照射灭活作用中的应用。这里描述的方法使用了一种针对几丁质孢子壁的荧光增白剂——荧光白，来观察发育中的孢子群，以确认感染性。将孢子悬液的系列稀释液接种到含有RK - 13细胞的组织培养孔载玻片上。然后冲洗载玻片，用甲醇固定，并用荧光白染色，进行显微镜检查。在感染的细胞单层上很容易看到大量发育中的孢子。在每个稀释步骤中，用阳性和阴性孔，通过最大可能数（MPN）统计分析来定量原始悬液中感染性孢子的数量。该测定法用于评估紫外线对水中肠脑炎微孢子虫孢子的消毒潜力。确定使感染性孢子数量减少3个对数（10）或99.9%所需的紫外线剂量为8.43 mW·s/cm²。
