Detection of UV-induced thymine dimers in individual Cryptosporidium parvum and Cryptosporidium hominis oocysts by immunofluorescence microscopy
Detection of UV-induced thymine dimers in individual Cryptosporidium parvum and Cryptosporidium hominis oocysts by immunofluorescence microscopy
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DOI：
10.1128/aem.01251-06
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发表时间：
2007-02-01
影响因子：
4.4
通讯作者：
Smith, H. V.
中科院分区：
文献类型：
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作者：
Al-Adhami, B. H.;Nichols, R. A. B.;Smith, H. V.
为了研究紫外线对体外培养的微小隐孢子虫和人隐孢子虫卵囊的影响，我们将完整卵囊暴露于4-、10-、20-和40-MJ。Cm(-2)剂量的紫外线照射。用抗环丁基胸腺嘧啶二聚体的单抗(抗TDmAb)在免疫荧光显微镜下检测胸腺嘧啶二聚体。通过与核荧光素4‘，6’-二氨基-2-苯基吲哚(DAPI)的共定位，证实了子孢子核内有二聚体特异的荧光。用商品异硫氰酸荧光素标记的抗隐孢子虫卵囊抗体(FITC-CMAB)或德克萨斯红标记的抗隐孢子虫卵囊抗体(TR-CMAB)显示卵囊壁。FITC-CMAB的使用在剂量低于40mJ时干扰TD的检测。Cm(-2)。联合使用抗TDmAb、tr-CMAB和DAPI，可在10~40mJ的卵囊内的子孢子核中检测到二聚体特异性荧光。厘米(-2)的紫外光。人毛滴虫也得到了类似的结果。微小隐孢子虫卵囊暴露于10~40MJ。厘米(-2)的紫外线未能感染新生小鼠，证实了我们的抗TD免疫荧光试验的结果与我们的新生小鼠传染性试验的结果是平行的。这些结果表明，我们的免疫荧光检测方法适用于检测紫外线照射引起的微小弧菌和人弓形虫卵囊的DNA损伤。
To investigate the effect of UV light on Cryptosporidium parvum and Cryptosporidium hominis oocysts in vitro, we exposed intact oocysts to 4-, 10-, 20-, and 40-mJ. cm(-2) doses of UV irradiation. Thymine dimers were detected by immunofluorescence microscopy using a monoclonal antibody against cyclobutyl thymine dimers (anti-TDmAb). Dimer-specific fluorescence within sporozoite nuclei was confirmed by colocalization with the nuclear fluorogen 4',6'-diamidino-2-phenylindole (DAPI). Oocyst walls were visualized using either commercial fluorescein isothiocyanate-labeled anti-Cryptosporidium oocyst antibodies (FITC-CmAb) or Texas Red-labeled anti-Cryptosporidium oocyst antibodies (TR-CmAb). The use of FITC-CmAb interfered with TD detection at doses below 40 mJ . cm(-2). With the combination of anti-TDmAb, TR-CmAb, and DAPI, dimer-specific fluorescence was detected in sporozoite nuclei within oocysts exposed to 10 to 40 mJ . cm(-2) of UV light. Similar results were obtained with C. hominis. C. parvum oocysts exposed to 10 to 40 mJ . cm(-2) of UV light failed to infect neonatal mice, confirming that results of our anti-TD immunofluorescence assay paralleled the outcomes of our neonatal mouse infectivity assay. These results suggest that our immunofluorescence assay is suitable for detecting DNA damage in C. parvum and C. hominis oocysts induced following exposure to UV light.