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Applied and Environmental Microbiology, February 2007, p. 947-955, Vol. 73, No. 3
0099-2240/07/$08.00+0     doi:10.1128/AEM.01251-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Detection of UV-Induced Thymine Dimers in Individual Cryptosporidium parvum and Cryptosporidium hominis Oocysts by Immunofluorescence Microscopy{triangledown}

B. H. Al-Adhami,1 R. A. B. Nichols,1 J. R. Kusel,2 J. O'Grady,3 and H. V. Smith1*

Scottish Parasite Diagnostic Laboratory, Stobhill Hospital, Glasgow G21 3UW, United Kingdom,1 Division of Infection & Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom,2 Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow G4 0NR, United Kingdom3

Received 31 May 2006/ Accepted 25 September 2006

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.

* Corresponding author. Mailing address: Scottish Parasite Diagnostic Laboratory, Stobhill Hospital, Glasgow G21 3UW, United Kingdom. Phone: 44 141-201-3028. Fax: 44 141-201-3029. E-mail: huw.smith{at}northglasgow.scot.nhs.uk.

{triangledown} Published ahead of print on 29 September 2006.

Applied and Environmental Microbiology, February 2007, p. 947-955, Vol. 73, No. 3
0099-2240/07/$08.00+0     doi:10.1128/AEM.01251-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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