- Email: [email protected]
Inactivation of Cryptosporidium parvum Oocysts by Low Pressure UV-light
Cryptosporidium: From Molecules to Disease R.C.A. Thompson, A. Armson and U.M. Ryan (Editors) © 2003 Published by Elsevier B.V. All rights reserved
261
Chapter 33
Inactivation of Cryptosporidium parvum Oocysts by Low Pressure UV-Light P. Karanis, D. Schoenen and O. Hoyer
Conventional treatment processes and chemical disinfection of water are not effective enough for the removal and inactivation of Cryptosporidium parvum oocysts from large water supplies. One of the most promising alternative methods for the oocyst inactivation is the ultraviolet (UV) irradiation of drinking water. We describe here inactivation of C parvum oocysts by lowpressure UV and the infectivity assay of low concentrations of C. parvum oocysts not exposed to UV-Hght. C parvum oocysts were obtained from fecal samples of infected cattle and purified by discontinuous sucrose gradients. Oocysts supplied for the UV inactivation experiments were 4 6 weeks old. After suspension into deionized water, oocysts were exposed at well-defined dosages of UV light (254 nm). Experiments were carried out under laboratory conditions with a specially constructed apparatus. We used 1 x 1 0 ^ oocysts/mL in a total volume of 30 mL in petri dishes and four selected UV dosages (300, 150, 75 and 30 J/m^). The efficacy of UV irradiation for oocyst inactivation was examined by in vivo infectivity assay using the SCID-mice model. SCID-mice (CB-l-17-scid/IcrCrl, male and female, 4 - 6 weeks old) were divided into different groups after delivery. Mice of the same group were orally administered an aHquot of 0.5 mL of the appropriate inoculum from the UV-exposed sample. As controls, mice of one group were inoculated with distilled water. In parallel, different mice groups were inoculated with oocysts not exposed to UV-light. This assay was used to determine the capacity of infectivity of low concentrations of C parvum oocysts. All animals were transferred into cages and kept separately after the inoculation process. Fecal samples were collected from all mice 5-18 days postinoculation (dpi) and diluted into distilled water. Oocyts from feces were purified by discontinuous sucrose gradients, stained by the Immunofluorescence test (IF test) and scanned by immunofluorescence microscopy for detection of oocysts. The actual selected and applied UV dosages, the actual oocyst inocula administered and their resultant percentage infection are summarized in Table 33.1. The actual oocyst numbers not exposed to UV and administered to mice and their resultant percentage infection are sunmiarized in Table 33.2. CONCLUSION UV-irradiation has a high potential for disinfection against Cryptosporidium by drinking water preparation. Cryptosporidium required the application of 300 J/m^ or lower UV doses for inactivation. Same oocyst inocula prepared from suspension of different dose UV-treated oocysts demonstrated same level of infectivity. For example irradiation with UV doses of 75 and
262 TABLE 33.1 Cryptosporidium infectivity experiments in SCID-mice irradiated oocysts—summary results Animal group 300 J/nf II III IV V 150 J/m^ VI VII VIII 75 J/m^ IX X 30 J/m^ XI
Number of inoculated oocysts
Number of animals positive/inoculated
% infectivity
1 X 10^ 1 X lO'* 1 X 10' 2.5 X 10'
0/10 0/6 0/15 1/10
0.0 0.0 0.0 10.0
2X 10' 2.5 X 10' 5X 10'
20/20 6/18 0/10
100.0 33.3 0.0
2X 10^ 4.5 X 10'
0/10 6/20
0.0 30.0
4.5 X 10'
7/20
35.0
30 J/m^ resulted in 30-35% infectivity of the inoculated animals. Although this phenomenon may be attributable in to variation in oocyst enumeration, the number of inoculated animals or individual mouse responses, we expect 4-log inactivation of C. parvum oocysts within these dose ranges. The in vivo infectivity assay is a reliable criterion to evaluate these experiments. Further studies with different Cryptosporidium isolates should be done in order to find out if different isolates of Cryptosporidium, have the same UV sensitivity as has been shown here. Use of different strains of C parvum oocysts can yield variability in the infectivity and inactivation experiments because of the high degree of biological variability of the parasites, but most probably this can affect the inactivation results only by low range of applied doses. In summary, these experiments and the results from previous studies (Karanis et al., 1992; Bukhari et al., 1999; Clancy et al., 2000) demonstrate that UV irradiation has the potential for effective inactivation of oocysts for disinfection purposes. The SCID-mice model (Karanis and Schoenen, 2001) indicates a reliable model for infectivity assays and is useful for application to similar situations with low numbers of potentially infectious oocysts purified from natural water samples, or to evaluate the efficiency of disinfection and inactivation of oocysts. TABLE 33.2 Cryptosporidium infectivity experiments in SCID-mice non-irradiated oocysts—sunmiary results Animal group
Number of inoculated oocysts
Number of animals positive/inoculated
% infectivity
I XII XIII XIV XV XVI XVII
0 1 1 1 1 1 1
0/34 1/31 3/36 4/34 22/35 28/34 35/37
0.0 3.2 8.3 11.8 62.9 82.4 94.6
X X X X X X
10" 10° 10' 10^ 10^ 10"^
263 REFERENCES Bukhari, Z., Hargy, T., Bolton, J., Dussert, B. and Clancy, J., 1999. Medium-pressure UV for oocyst inactivation. J. AWWA, 91: 86-94. Clancy, J.L., Bukhari, Z., Hargy, T.M., Bolton, J.R., Dussert, B.W. and Marshall, M.M., 2000. Using UV to inactivate Cryptosporidium. J. AWWA, 92: 97-104. Karanis, P., Maier, W.A., Schoenen, D. and Seitz, H.M., 1992. UV sensitivity of protozoan parasites. J. Water SRTAqua, 41:95-100. Karanis, P. and Schoenen, D., 2001. Biological test for the detection of low concentrations of infectious Cryptosporidium parvum oocysts in water. Acta Hydrochim. Hydrobiol., 29: 1-4.
261
Chapter 33
Inactivation of Cryptosporidium parvum Oocysts by Low Pressure UV-Light P. Karanis, D. Schoenen and O. Hoyer
Conventional treatment processes and chemical disinfection of water are not effective enough for the removal and inactivation of Cryptosporidium parvum oocysts from large water supplies. One of the most promising alternative methods for the oocyst inactivation is the ultraviolet (UV) irradiation of drinking water. We describe here inactivation of C parvum oocysts by lowpressure UV and the infectivity assay of low concentrations of C. parvum oocysts not exposed to UV-Hght. C parvum oocysts were obtained from fecal samples of infected cattle and purified by discontinuous sucrose gradients. Oocysts supplied for the UV inactivation experiments were 4 6 weeks old. After suspension into deionized water, oocysts were exposed at well-defined dosages of UV light (254 nm). Experiments were carried out under laboratory conditions with a specially constructed apparatus. We used 1 x 1 0 ^ oocysts/mL in a total volume of 30 mL in petri dishes and four selected UV dosages (300, 150, 75 and 30 J/m^). The efficacy of UV irradiation for oocyst inactivation was examined by in vivo infectivity assay using the SCID-mice model. SCID-mice (CB-l-17-scid/IcrCrl, male and female, 4 - 6 weeks old) were divided into different groups after delivery. Mice of the same group were orally administered an aHquot of 0.5 mL of the appropriate inoculum from the UV-exposed sample. As controls, mice of one group were inoculated with distilled water. In parallel, different mice groups were inoculated with oocysts not exposed to UV-light. This assay was used to determine the capacity of infectivity of low concentrations of C parvum oocysts. All animals were transferred into cages and kept separately after the inoculation process. Fecal samples were collected from all mice 5-18 days postinoculation (dpi) and diluted into distilled water. Oocyts from feces were purified by discontinuous sucrose gradients, stained by the Immunofluorescence test (IF test) and scanned by immunofluorescence microscopy for detection of oocysts. The actual selected and applied UV dosages, the actual oocyst inocula administered and their resultant percentage infection are summarized in Table 33.1. The actual oocyst numbers not exposed to UV and administered to mice and their resultant percentage infection are sunmiarized in Table 33.2. CONCLUSION UV-irradiation has a high potential for disinfection against Cryptosporidium by drinking water preparation. Cryptosporidium required the application of 300 J/m^ or lower UV doses for inactivation. Same oocyst inocula prepared from suspension of different dose UV-treated oocysts demonstrated same level of infectivity. For example irradiation with UV doses of 75 and
262 TABLE 33.1 Cryptosporidium infectivity experiments in SCID-mice irradiated oocysts—summary results Animal group 300 J/nf II III IV V 150 J/m^ VI VII VIII 75 J/m^ IX X 30 J/m^ XI
Number of inoculated oocysts
Number of animals positive/inoculated
% infectivity
1 X 10^ 1 X lO'* 1 X 10' 2.5 X 10'
0/10 0/6 0/15 1/10
0.0 0.0 0.0 10.0
2X 10' 2.5 X 10' 5X 10'
20/20 6/18 0/10
100.0 33.3 0.0
2X 10^ 4.5 X 10'
0/10 6/20
0.0 30.0
4.5 X 10'
7/20
35.0
30 J/m^ resulted in 30-35% infectivity of the inoculated animals. Although this phenomenon may be attributable in to variation in oocyst enumeration, the number of inoculated animals or individual mouse responses, we expect 4-log inactivation of C. parvum oocysts within these dose ranges. The in vivo infectivity assay is a reliable criterion to evaluate these experiments. Further studies with different Cryptosporidium isolates should be done in order to find out if different isolates of Cryptosporidium, have the same UV sensitivity as has been shown here. Use of different strains of C parvum oocysts can yield variability in the infectivity and inactivation experiments because of the high degree of biological variability of the parasites, but most probably this can affect the inactivation results only by low range of applied doses. In summary, these experiments and the results from previous studies (Karanis et al., 1992; Bukhari et al., 1999; Clancy et al., 2000) demonstrate that UV irradiation has the potential for effective inactivation of oocysts for disinfection purposes. The SCID-mice model (Karanis and Schoenen, 2001) indicates a reliable model for infectivity assays and is useful for application to similar situations with low numbers of potentially infectious oocysts purified from natural water samples, or to evaluate the efficiency of disinfection and inactivation of oocysts. TABLE 33.2 Cryptosporidium infectivity experiments in SCID-mice non-irradiated oocysts—sunmiary results Animal group
Number of inoculated oocysts
Number of animals positive/inoculated
% infectivity
I XII XIII XIV XV XVI XVII
0 1 1 1 1 1 1
0/34 1/31 3/36 4/34 22/35 28/34 35/37
0.0 3.2 8.3 11.8 62.9 82.4 94.6
X X X X X X
10" 10° 10' 10^ 10^ 10"^
263 REFERENCES Bukhari, Z., Hargy, T., Bolton, J., Dussert, B. and Clancy, J., 1999. Medium-pressure UV for oocyst inactivation. J. AWWA, 91: 86-94. Clancy, J.L., Bukhari, Z., Hargy, T.M., Bolton, J.R., Dussert, B.W. and Marshall, M.M., 2000. Using UV to inactivate Cryptosporidium. J. AWWA, 92: 97-104. Karanis, P., Maier, W.A., Schoenen, D. and Seitz, H.M., 1992. UV sensitivity of protozoan parasites. J. Water SRTAqua, 41:95-100. Karanis, P. and Schoenen, D., 2001. Biological test for the detection of low concentrations of infectious Cryptosporidium parvum oocysts in water. Acta Hydrochim. Hydrobiol., 29: 1-4.