Efficiancy of UV light in Inactivating Cryptosporidium parvum Oocysts

1. Efficiancy of UV light in Inactivating Cryptosporidium parvum Oocysts Morita S, Namikoshi A, Hirata T, Oguma K, Katayama H, Ohgaki S, Motoyama N, Fujiwara M. Applied Environmental Microbiology 2002 Nov;68(11):5387-93

2. What is UV light?

4. UV

6. Background Cryptosporidium parvum (zoonotic parasite) Diarrhea (not bloody) Very infective Infects all ages Immunity acuired Resistant oocysts No good cure Transmitable via other mammals Dangerous for immunocompromised mammals Can be dangerous in combination with other diseases

7. Background Problem Cryptosporidium parvum common in surface water/rivers C. parvum resistant to normal water treatment (chlorine) Mineral water must be pathogen free

8. Background Solution – UV water treatment No/little hazard Easy to maintain Good results with virus and bacteria Inactivates virus and bacteria (DNA)

9. Background Problems with UV water treatment DNA repair Photoreactivation (enzyme lesion repair by near UV-light) Dark Repair (repair in absence of light) Previous Studies: In Vitro excystation & Vital Dye Methods No inactivation of C. parvum

10. Background

11. Materials& Methods Treatment + SCID C. parvum oocysts Purified C. parvum oocysts

12. Materials& Methods Summary Experimental Conditions Table 1 UV dose (mWs/cm2) 0 1.2 1.68 1.80 1.82 2.40 40 80 120 160 240 + + + - - + - - - - - T (50C) T (100C) T (200C) T (300C) + + + - - + - - - - - + + - + + + + + + + + + + + - - + - - - - - Animal Infection In Vitro Excystation

13. Materials& Methods UV treatment and Recovery Table 2 200C and 0.10 mW/cm2 UV dose (mWs/cm2) 0.5 1.0 1.5 Evaluation FL dose (mWs/cm2) 150 X X 180 X 300 X X 360 X 540 X 720 X Storage (h) 4 X 12 X X X 24 X X X Not Treated X X X Animal Infection (AI) & Endonuclease-sensitive site asssay (ESS)

14. Materials& Methods Animal Infection Increasing infection dose (oocysts) (n=5-8 mice/group) Relatice Risk of infection

15. Materials& Methods In Vitro Excystation Excystation rate (V) = IO = Intact Oocysts (prior excystation) S = free Sporocysts PO = Partially excysted Oocysts EO = Empty Oocysts VV Survival Rate (Sr) = V = Excysting rate (UV irradiated) V = Excysting rate (control)

16. Results Excystation Metod Oocysts Preincubation: Hanks balanced salt solution, 5 min Woodmansee DB 1987 Oocysts Sodium hyochlorite Taurocholic acid solutions, 7 pH Incubation 370C, 60 min

17. Results Figure 1: UV dose vs. Infectivity (mice) ~ ½ dose needed for infectivity reduction according to litterature (16 mWs/cm2 recommended for 2 log10 reduction) 1 log10 reduction 2 log10 reduction 4 log10 reduction

18. Results Figure 2: UV dose vs. Survival (oocysts) 230 mWs/cm2 dose needed for 2 log10 reduction in viability ...dose 200 time higher than reduction in infectivity (1 mWs/cm2) Contact time (CT) for Chlorine 18 times higher for viability reduction Contact time (CT) for Ozone 3 times higher for viability reduction Conlusion: oocyst excyst but do not infect with tested doses

19. Results Figure 3: UV dose vs. Relative Infectivity (mice) Dose increase for 2 log10 reductionin infectivity was 7% at 100C decrease UV as disinfectant farily stable at normal temperatures (in contrast to eg. Ozone) ■ 5 0C □ 10 0C ● 30 0C Water temperature 2 log10 reduction (mWs/cm2): 1.20 1.07 1.02

20. Results Figure 4: UV dose vs. Relative Infectivity (mice) UV Dose for 2 log10reductionin infectivity was: 1.15 mWs/cm2 ...for0.048 mW/cm2 1.20 mWs/cm2...for0.12 mW/cm2 1.34 mWs/cm2...for0.60 mW/cm2 8% UV dose increase  10 fold increase in intensity Conclusion figure 3+4: UV irradiation can inactivate C. parvum without regard to water temperature and intensity ■ 0.048 mW/cm2□ 0.12 mW/cm2● 0.60 mW/cm2

21. Results Figure 5: Fluorescent light dose vs. ESS (DNA) Increased UV dose induced pyrimidine dimers repaired with increased exposure time to fluorescent light 30-50% reduction in ESS at 750 mWs/cm2 dose ■ 0.5 mWs/cm2□ 1.0 mWs/cm2

22. Results Figure 6: Dark Storage vs. ESS (DNA) UV induced pyrimidine dimers were repaired with dark storage periods Dark storage recovery was slower than fluorecent light exposure 60% ESS repair in 24 hours Conclusion figure 5+6: Both fluorecent light exposure and dark storage can repair UV irradiated damage to C. parvumoocyst ■ 0.5 mWs/cm2□ 1.0 mWs/cm2

23. Results Infectivity of fluorecent irradiated reactived oocysts UV irradiation with... 0.50 mWs/cm2 led to0.98 log10 reduction in infectivity 1.00 mWs/cm2led to2.00 log10 reduction in infectivity 1.50 mWs/cm2led to3.15 log10 reduction in infectivity ...but no change in infectivity when reactivated with fluorecent irradiatiation

24. Results Infectivity of dark storage reactived oocysts No change in infectivity when reactivated in dark storage for 4 and 24 hours Conclusion: UV irradiation of oocysts does not recover infectivity though DNA damage is documented I) Dark repair occurs parralel with fluorecent light reactivation II) Remains of DNA damage still affect infectivity III) Unknow lesions on DNA inhibits infectivity

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