Open AccessDetection of UV-Induced Thymine Dimers in Individual Cryptosporidium parvum and Cryptosporidium hominis Oocysts by Immunofluorescence Microscopy
Author(s) -
Batol Al-Adhami,
R.A.B. Nichols,
J. R. Kusel,
J. E. O'grady,
H. V. Smith
Publication year - 2007
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.01251-06
Subject(s) - cryptosporidium parvum , cryptosporidium , dapi , immunofluorescence , biology , pyrimidine dimer , microbiology and biotechnology , fluorescence microscope , staining , virology , thymine , antibody , fluorescence , dna , dna damage , biochemistry , genetics , physics , quantum mechanics , immunology , feces
To investigate the effect of UV light onCryptosporidium parvum andCryptosporidium 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 withC. 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 inC. parvum andC. hominis oocysts induced following exposure to UV light.