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Inactivation of oocysts of Cryptosporidium parvum by ultraviolet irradiation
~
Pergamon
0043-1354(95)00072-0
War. Res. Vol. 29, No. I1, pp. 2583-2586, 1995 Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0043-1354/95 $9.50 + 0.00
RESEARCH NOTE I N A C T I V A T I O N OF OOCYSTS OF C R Y P T O S P O R I D I U M P A R V U M BY U L T R A V I O L E T I R R A D I A T I O N A. T. C A M P B E L L , l L. J. R O B E R T S O N , 1'2 M. R. S N O W B A L L 3 a n d H. V. S M I T H l ~Scottish Parasite Diagnostic Laboratory, Stobhill NHS Trust, Springburn, Glasgow G21 3UW, 2Division of Environmental Health, Department of Civil Engineering, University of Strathclyde, Glasgow, Scotland and 3Water Recovery plc, Oxford OX6 9LF, England (First received March 1995; accepted March 1995) Abstract--Inactivation of oocysts of Cryptosporidium parvum in clean water using a novel design of an ultraviolet disinfection system was assessed by a vital dye assay and by in vitro excystation. The disinfection unit system is designed to expose the oocysts to ultraviolet radiation on two filters, providing a maximum total exposure to ultraviolet radiation of 8748 mW s cm -2. Results revealed a reduction in oocyst viability of over two logs, indicating that this treatment has exciting potential as an additional treatment for water already treated by conventional methods. However, these data are only preliminary results using one isolate of oocysts and further trials must be conducted before this system could be recommended for use. Key words--Cryptosporidium, disinfection, oocysts, water treatment, ultraviolet, viability
INTRODUCTION A n u m b e r o f factors intrinsic to the biology o f Cryptosporidium p a r v u m facilitate the transmission o f this parasite via the w a t e r b o r n e route, including its r o b u s t nature, small size, the small infective dose a n d the large n u m b e r o f oocysts excreted by an infected individual ( R o b e r t s o n et al., 1994). A wide-spread low level c o n t a m i n a t i o n o f surface waters with oocysts has been described for b o t h E u r o p e a n d U S A (Ongerth a n d Stibbs, 1987; LeChevallier et al., 199t; N a t i o n a l C r y p t o s p o r i d i u m Survey G r o u p , 1992; D e L e o n et al., 1993) a n d a n u m b e r of o u t b r e a k s of w a t e r b o r n e cryptosporidiosis have occurred, perhaps the most n o t a b l e o f which was the o u t - b r e a k in Milwaukee, U.S.A. in 1993 in which over 400,000 individuals were infected ( M a c K e n z i e et al., 1994). As at least three o u t b r e a k s have been attributed to oocysts in drinking water supplies in which multiple barriers were used in water treatment, current water t r e a t m e n t processes are clearly n o t entirely a d e q u a t e in removing or inactivating oocysts o f C. p a r v u m ( R o b e r t s o n et al., 1994). The use o f chlorine as a final disinfectant in water t r e a t m e n t has become virtually ubiquitous, however C. p a r v u m oocysts are insensitive to the c o n c e n t r a t i o n s routinely used (Smith et al., 1989; Korich et al., 1990) a n d thus there is e n o r m o u s interest in the d e v e l o p m e n t of an alternative, m o r e effective disinfectant which does n o t result in b y - p r o d u c t s potentially d a m a g i n g to h u m a n health. Results published in the scientific literature indicate that, to date, ozone is p r o b a b l y the most effective disinfectant, followed by chlorine dioxide
which is, perhaps, one fifth as effective. O t h e r disinfectants t h a t have been tested, including ultraviolet (u.v.), have been considered to be incapable o f effective inactivation at practical levels of application. F o r example, L o r e n z o - L o r e n z o et al. (1993) describe inactivation of oocysts (as assessed by infectivity) only after a prolonged period (150 min) in a u.v. disinfection system. However the data in this study are difficult to interpret as the irradiation dose is not stated and the dose c a n n o t be calculated from the data described. In a n o t h e r study, R a n s o m e et al. (1993) f o u n d t h a t exposure to u.v. light only produced 9 0 % inactivation at 8 0 m W s c m 2 a n d 9 9 % inactivation at 120 m W s cm -2. A n y novel t r e a t m e n t or novel application of treatm e n t which has the potential to inactivate oocysts must thus be of interest. However, rigorous testing is imperative a n d the procedure m u s t be f o u n d to be wholly reliable, a n d the p r o d u c t i o n o f by-products which m a y be potentially d a m a g i n g to h u m a n health must be investigated, before it can be r e c o m m e n d e d for use. In this note we describe preliminary results which indicate the potential for inactivation of oocysts of C. p a r v u m by u.v. light using a novel design of disinfection unit.
METHODS Source and purification o f oocysts Oocysts of C. parvum were obtained from bovine faeces from a natural infection and were purified by sucrose and Percoll flotation. Purified oocysts were stored at 4~C in water.
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U.V. disinfection unit The u.v. disinfection unit is specifically designed for the purposes of inactivating Cryptosporidium and Giardia in clean water (Fig. 1). In brief, the unit consists of two treatment chambers each containing a 2/zm nominal filter. O n to each side of each filter are focused 3 LP Mercury A R C lamps (i.e. 6 lamps per filter), giving a theoretical m i n i m u m radiation intensity of approximately 14.58 m W cm 2, at the germicidal wavelength of 253.7 nm. The system is designed to capture any cysts or oocysts in the water on the first filter, then the flow within the filter chamber is reversed thereby back-flushing cysts or oocysts on to the second filter where they are trapped until a pre-set u.v. dose is reached. By the use of 8 valves, the pattern of water flow is designed to ensure that any cysts and oocysts will be captured on both membranes, but there will be no build up of debris within the system. The captured cysts or oocysts are thus exposed to a pre-set u.v. irradiation dose, totally independent of the flow rate. Flow rate through the system is designed to be approximately 2 0 0 m 3 h ~ when in use on-line. For these experiments the oocysts were trapped on each filter for a m a x i m u m of five minutes thereby exposing them to u.v. irradiation of approximately 8748 m W s cm 2, and the flow rate was set to be approximately 10 m 3 h ~. Experimental design Viability of oocysts was assessed by morphology and inclusion/exclusion of vital dyes (Campbell et al., 1992) before use and excysted according to Robertson et al. (1993). The excystation was examined after 30 min to ensure that motile sporozoites were observed. In experiments, conducted in triplicate, 5 × 107 oocysts in 2.5 ml water were added to the disinfection unit (equivalent to approximately I05 oocysts/l) and the u.v. mechanism switched on for 5 min on each of the two filters. Water in the unit was pre-filtered through a 1/~m yarn filter to minimise the presence of extraneous debris. For each run conducted with the u.v. mechanism switched on, a control experiment was run first in which the water and oocysts were pumped around the machine for 20 min but the u.v. mechanism was not switched on and valves were kept open to minimise flow through the machine's filters. Oocysts were then collected from the machine and their viability assessed. Temperature and pH were measured during each of the experiments.
Concentration o f oocysts from the disinfection unit To concentrate oocysts from the disinfection unit (total volume approx. 5001), a water sample (251) was drained from the purpose built side-arm of the machine sited immediately after the second filter/valve assembly. In the experimental runs, this was timed to capture a representative sample following the backflush of the oocysts from the filter. Samples obtained were then pumped through a 142 m m Sartorius filter (0.2/~m, nylon) and the filter cut up and washed in 200 ml 0.1% Tween-80 solution. This was then concentrated by a series of centrifugation steps at 1060g to give a final volume of 1 ml. Assessment o f ooeysts viability using the vital dye assay Viability of oocysts was assessed using the vital dye assay of Campbell et al. (1992) which relies upon morphology and inclusion/exclusion of two vital dyes, propidium iodide (PI) and 4'6 diamidino-2-phenyl indole (DAPI). A I h pre-incubation in acidified HBSS (Hanks' Balanced Salt Solution) (pH 2.75) was conducted prior to the incubation with the vital dyes in order to maximise vital dye uptake. In the final 30 min of incubation with the dyes, fluorescein isothiocyanate-labelled anti-Cryptosporidium monoclonal antibody was added to assist in the identification of the oocysts. At least 200 oocysts were assessed for viability in each trial and control.
Assessment o f viability using excystation Viability was also assessed by in vitro excystation of oocysts following the procedure of Robertson et al. (1993). Following a pre-incubation in acidified HBSS in order to maximise excystation, the oocysts were incubated in bile and sodium hydrogen carbonate in Hanks' Minimal Essential Medium. After 30 min and 4 h, aliquots of the excystation incubation were assessed by microscopy with enumeration of empty oocyst shells, non-excysted oocysts, partly excysted oocysts and motile and non-motile sporozoites. In trials 2 and 3 the aliquot taken after 30 min in excystation medium was examined in total by reading the whole sample contained beneath a 22 × 22 m m cover slip. Microscopy All microscopy was conducted on an Olympus BH-2 fluorescence microscope equipped with appropriate filter blocks for visualisation o f the dyes and with Nomarski
Fig. 1. Ultraviolet disinfection unit showing the two filtration/u.v, chambers.
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Table 1. Viability of control and experimental trial C. parvum oocysts by vital dye assay % non-viable Number of % viable (DAPI+/PI+ or % potentially oocysts (DAPI +/PI - ) empty shells) viable (DAPI -/PI - ) counted Control I 84 15 1 926 Control 2 86 14 -230 Control 3 89 11 -209 Trial 1 -100 -1164 Trial 2 -100 -216 Trial 3 -100 -211
optics for improved visualisation of the morphology for enumeration of the excystation.
DISCUSSION
Oocyst viability was considered to between 87 a n d 8 9 % before the trials were c o n d u c t e d with between 10 a n d I 1% o f the oocysts non-viable. The results o f the trials a n d controls are described in Tables 1-3. The viability o f the control oocysts was not significantly different to the initial viability before the oocysts were i n t r o d u c e d into the disinfection unit. Circulating the oocysts t h r o u g h the unit w i t h o u t the u.v. light a n d the subsequent c o n c e n t r a t i o n technique did not affect oocyst viability. D u r i n g the three trials the t e m p e r a t u r e of the water in the unit varied between 14-19°C a n d the p H varied between 7.9 a n d 8.2. A good correlation between the vital dye assay a n d the maximised 4 h excystation was observed; m e a n excystation in the control g r o u p was 9 0 % a n d the expected viability from the vital dye assay was 86%. Sporozoite ratio in the control samples was high after 30 min, but reduced after 4 h, p r e s u m a b l y due to lysis of sporozoites ( R o b e r t s o n et al. 1993). M o s t ( > 9 9 % ) free sporozoites observed in the excystation media were motile. Oocysts in the experimental samples also h a d their viability assessed by b o t h the vital dye assay a n d in vitro excystation. The vital dye assay indicated t h a t oocyst viability h a d been reduced by over 2logs, with no viable or potentially-viable oocysts observed. In vitro excystation confirmed the results of the vital dye assay, with no excystation observed. Free sporozoites were not observed after either 30 min or 4 h in the excystation media, even when the whole aliquot of the 3 0 m i n excystation (equivalent to approx. 10 s oocysts) b e n e a t h the coverslip was examined.
A l t h o u g h purification of water by u.v. t r e a t m e n t has been u n d e r consideration for several years, only in recent years has it been seriously considered as an option for potable water supplies (Snowball a n d Hornsey, 1988; Wolfe, 1990). However, the resistance of b o t h cysts of Giardia a n d the m o r e robust oocysts of C r y p t o s p o r i d i u m to u.v. systems tested has, to date, indicated t h a t u.v. t r e a t m e n t is unsuitable for treatm e n t of potable water supplies. These preliminary results indicate t h a t with the u.v. disinfection system used in this study there is the potential for inactivating oocysts o f C. p a r v u m in clean water. A l t h o u g h e n u m e r a t i o n of the experiment described here indicated between 2 a n d 3 l o g s reduction in viability of the oocysts (99.9% inactivation), it should be n o t e d that this figure is restricted by the limits of the s t a n d a r d e n u m e r a t i o n technique; extended e n u m e r a t i o n might be speculated to reveal the actual log reduction in viability to be greater t h a n this. The best t r e a t m e n t expected at present from a single procedure in water t r e a t m e n t is considered to be 3 log scale ( R o b e r t s o n et al. 1994), and these results indicate t h a t this disinfection system has the potential to match, or perhaps to exceed this figure. A multi-barrier a p p r o a c h has been considered the most practical a p p r o a c h to water t r e a t m e n t (Robertson et al. 1994) a n d this disinfection system, designed for treated water, m a y c o n t r i b u t e a valuable additional barrier. The t e m p e r a t u r e a n d p H at which these experiments were conducted would not be considered to have h a d any impact on the viability of the oocysts ( R o b e r t s o n et al. 1992; Fayer, 1994). Similar experiments have also been conducted o n the inactivation of a range of bacterial a n d viral cultures with a similar, a l t h o u g h not identical, u.v. disinfection unit (Snowball, M. R.; unpublished data). In these experiments, log kills have been
Table 2. Excystation of C. parvum oocysts in control and experimental trials after 30 mins in excystation medium Number of oocysts Excystation (%) Sporozoite ratio counted Control 1 66 3.8 174 Control 2 59 3.7 116 Control 3 30 4.2 154 Trial 1 --200 Trial 2 --200 Trial 3 --200
Table 3. Excystation of C. parvum oocysts in control and experimental trials after 4 hs in excystation medium Number of oocysts Excystation (%) Sporozoiteratio counted Control 1 89 0.2 208 Control 2 89 0.5 206 Control 3 92 0.4 209 Trial I --208 Trial 2 --218 Trial 3 --252
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Research Note
measured between 3 (Legionella spiritensis) to 7.2 (Salmonella typhimurium). These kills were considered to be in excess of those published for other u.v. disinfection systems with no p h o t o r e a c t i v a t i o n or resuscitation. CONCLUSIONS 1. These preliminary data suggest that the u.v. disinfection unit used for these experiments has the potential for inactivating oocysts of C. parvum in treated water, a n d thus m a y be of importance for inclusion "in-line" following conventional water treatment. 2. The 2-3 log reduction in oocyst viability suggested by the data presented in this note, is restricted by the limits o f the s t a n d a r d e n u m e r a t i o n technique; extended e n u m e r a t i o n by using a n autom a t e d c o u n t i n g system such as flow cytometry m i g h t be speculated to reveal the actual log reduction in viability to be greater t h a n this. 3. Whilst the a u t h o r s consider the data described in this c o m m u n i c a t i o n to be highly significant, it should be emphasised that the studies described represent only 3 trials with one isolate of oocysts and the following trials would also be r e c o m m e n d e d in order to verify these preliminary findings: (a) infectivity studies (b) experiments using different isolates of C. parvum, in particular with oocysts isolated from h u m a n infections, (c) studies with e n u m e r a t i o n of vital dye increased by several orders of m a g n i t u d e by using a n a u t o m a t e d c o u n t i n g system such as flow cytometry (d) Giardia trials: a l t h o u g h C. parvum oocysts are considered to be more r o b u s t t h a n Giardia cysts, it is i m p o r t a n t to test formally that Giardia cysts are also inactivated by this system. This is particularly pertinent as results from one study (Rice a n d Hoff, 1981) indicate that a u.v. dose of 63 m W s c m 2 resulted in between 7 0 - 8 0 % reduction in viability of Giardia cysts, while a n o t h e r group of workers ( R a n s o m e et al., 1993) report that the same u.v. dose applied in the same m a n n e r caused an 80% reduction in viability of C. parvum oocysts. A 9 0 % inactivation of Giardia muris cysts has also been reported (Carlson et al., 1990) at a u.v. dose o f 8 2 m W s c m 2. (e) Studies using a flow-through system in the o p e r a t i o n of the disinfection unit: in the experimental trials described here, the system used to facilitate sampling a n d c o n c e n t r a t i o n of oocysts required the water to be recirculated within the disinfection unit. This set-up restricted testing whether the 2 # m filter actually retained all the oocysts in a single pass. In order to ascertain that viable oocysts will not pass through the filter, a flow-through system should be assessed in further trials.
REFERENCES
Campbell A. T., Robertson L. J. and Smith H. V. (1992) Viability of Cryptosporidium parvum oocysts: correlation of in vitro excystation with inclusion/exclusion of fluorogenic vital dyes. Appl. environ. Microbiol. 58, 3488 3493. Carlson D. A. et al. (1985) Project summary: ultraviolet disinfection of water for small water supplies. Office of Research and Development, U.S. Environmental Protection Agency, Cincinnati, Ohio. EPA/600/S2-85/092. As quoted in Wolfe R. L. (1990). Ultraviolet disinfection of potable water: current technology and research needs. Environ. Svi. Technol 24, 768 773. DeLeon R., Rose J. B., Bosch A., Torrella F. and Gerba C. P. (1993) Detection of Giardia, Cryptosporidium and enteric viruses in surface and tap water samples in Spain. Int. J. environ. Hlth Res. 3, 121 129. Fayer R. (1994) Effect of high temperature on infectivity of Cryptosporidium parvum oocysts in water. Appl. environ. Microbiol. 60, 2732 2735. Korich D. G., Mead J. R., Madore M. S., Sinclair N. A, and Sterling C. R. (1990). Effects of ozone, chlorine dioxide, chlorine and monochloramine on Co,ptosporidium oocyst viability. Appl. environ. Microbiol. 56, 1423 1428. LeChevallier M. W., Norton W. D. and Lee R. G. (1991) Occurrence of Giardia and Cryptosporidium in surface water supplies. Appl. environ. Microbiol. 57, 2610-2616. Lorenzo-Lorenzo M. J., Ares-Mazas M. E., VillacortaMartinez de Maturana and Duran-Oreiro D. (1993) Effect of ultraviolet disinfection of drinking water on the viability of Cryptosporidium parvum oocysts. J. Parasitol. 79, 67-70. MacKenzie W. R., Hoxie N. J., Proctor M. E., Gradus M. S., Blair K. A., Peterson D. E., Kazmierczak J. J., Addiss D. G., Fox K. R., Rose J. B. and Davis J. P. (1994) A massive outbreak in Milwaukee of Crypto~poridium infection transmitted through the public water supply. N. Engl. J. Med. 331, 161 167. National Cryptosporidium Survey Group (1992) A survey of Cryptosporidium oocysts in surface and groundwaters in the UK. J. Inst. War. environ. Mgmt 6, 697-703. Ongerth J. E. and Stibbs H. H. (1987) Identification of Cryptay~oridium oocysts in river water. Appl. environ. Microbiol. 53, 672. Ransome M. E., Whitmore T. N. and Carrington E. G. (1993) Effect of disinfectants on the viability of Cryptosporidium parvum. Wat. Suppl. ll, Amsterdam, pp. 75-89. Rice E. W. and Hoff J. C. (1981) Inactivation of Giardia lamblia cysts by ultraviolet irradiation. Appl. environ. Microbiol. 42, 546 547. Robertson L. J., Campbell A. T. and Smith H. V. (1992) Survival of oocysts of Cryptosporidium parvum under various environmental pressures. Appl. environ. Microbiol. 58, 3494 3500. Robertson L. J., Campbell A. T. and Smith H. V. (1993) In vitro excystation of Cryptosporidiurn parvum. Parasitology 106, 13 19. Robertson L. J., Smith H. V. and Ongerth J. E. (1994) Cryptosporidium and cryptosporidiosis. Part III: Development of water treatment technologies to remove and inactivate oocysts. Microbiology Europe 2, 18 26. Smith H. V., Smith A. L., Girdwood R. W. A. and Carrington E. G. (1989) The effect of free chlorine on the viability of Cryptosporidium spp. oocysts. WRc, Bucks, U.K. Snowball M. R. and Hornsey I. S. (1988) Purification of water supplies using ultraviolet light. In Developments in Food Microbiology 3 (7) (Edited by Robinson R. K.), Vol. 3(7), pp. 171 191. Elsevier Applied Science. Wolfe R. L. (1990) Ultraviolet disinfection of potable water: current technology and research needs. Environ. Sci. Technol. 24, 768--773.
Pergamon
0043-1354(95)00072-0
War. Res. Vol. 29, No. I1, pp. 2583-2586, 1995 Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0043-1354/95 $9.50 + 0.00
RESEARCH NOTE I N A C T I V A T I O N OF OOCYSTS OF C R Y P T O S P O R I D I U M P A R V U M BY U L T R A V I O L E T I R R A D I A T I O N A. T. C A M P B E L L , l L. J. R O B E R T S O N , 1'2 M. R. S N O W B A L L 3 a n d H. V. S M I T H l ~Scottish Parasite Diagnostic Laboratory, Stobhill NHS Trust, Springburn, Glasgow G21 3UW, 2Division of Environmental Health, Department of Civil Engineering, University of Strathclyde, Glasgow, Scotland and 3Water Recovery plc, Oxford OX6 9LF, England (First received March 1995; accepted March 1995) Abstract--Inactivation of oocysts of Cryptosporidium parvum in clean water using a novel design of an ultraviolet disinfection system was assessed by a vital dye assay and by in vitro excystation. The disinfection unit system is designed to expose the oocysts to ultraviolet radiation on two filters, providing a maximum total exposure to ultraviolet radiation of 8748 mW s cm -2. Results revealed a reduction in oocyst viability of over two logs, indicating that this treatment has exciting potential as an additional treatment for water already treated by conventional methods. However, these data are only preliminary results using one isolate of oocysts and further trials must be conducted before this system could be recommended for use. Key words--Cryptosporidium, disinfection, oocysts, water treatment, ultraviolet, viability
INTRODUCTION A n u m b e r o f factors intrinsic to the biology o f Cryptosporidium p a r v u m facilitate the transmission o f this parasite via the w a t e r b o r n e route, including its r o b u s t nature, small size, the small infective dose a n d the large n u m b e r o f oocysts excreted by an infected individual ( R o b e r t s o n et al., 1994). A wide-spread low level c o n t a m i n a t i o n o f surface waters with oocysts has been described for b o t h E u r o p e a n d U S A (Ongerth a n d Stibbs, 1987; LeChevallier et al., 199t; N a t i o n a l C r y p t o s p o r i d i u m Survey G r o u p , 1992; D e L e o n et al., 1993) a n d a n u m b e r of o u t b r e a k s of w a t e r b o r n e cryptosporidiosis have occurred, perhaps the most n o t a b l e o f which was the o u t - b r e a k in Milwaukee, U.S.A. in 1993 in which over 400,000 individuals were infected ( M a c K e n z i e et al., 1994). As at least three o u t b r e a k s have been attributed to oocysts in drinking water supplies in which multiple barriers were used in water treatment, current water t r e a t m e n t processes are clearly n o t entirely a d e q u a t e in removing or inactivating oocysts o f C. p a r v u m ( R o b e r t s o n et al., 1994). The use o f chlorine as a final disinfectant in water t r e a t m e n t has become virtually ubiquitous, however C. p a r v u m oocysts are insensitive to the c o n c e n t r a t i o n s routinely used (Smith et al., 1989; Korich et al., 1990) a n d thus there is e n o r m o u s interest in the d e v e l o p m e n t of an alternative, m o r e effective disinfectant which does n o t result in b y - p r o d u c t s potentially d a m a g i n g to h u m a n health. Results published in the scientific literature indicate that, to date, ozone is p r o b a b l y the most effective disinfectant, followed by chlorine dioxide
which is, perhaps, one fifth as effective. O t h e r disinfectants t h a t have been tested, including ultraviolet (u.v.), have been considered to be incapable o f effective inactivation at practical levels of application. F o r example, L o r e n z o - L o r e n z o et al. (1993) describe inactivation of oocysts (as assessed by infectivity) only after a prolonged period (150 min) in a u.v. disinfection system. However the data in this study are difficult to interpret as the irradiation dose is not stated and the dose c a n n o t be calculated from the data described. In a n o t h e r study, R a n s o m e et al. (1993) f o u n d t h a t exposure to u.v. light only produced 9 0 % inactivation at 8 0 m W s c m 2 a n d 9 9 % inactivation at 120 m W s cm -2. A n y novel t r e a t m e n t or novel application of treatm e n t which has the potential to inactivate oocysts must thus be of interest. However, rigorous testing is imperative a n d the procedure m u s t be f o u n d to be wholly reliable, a n d the p r o d u c t i o n o f by-products which m a y be potentially d a m a g i n g to h u m a n health must be investigated, before it can be r e c o m m e n d e d for use. In this note we describe preliminary results which indicate the potential for inactivation of oocysts of C. p a r v u m by u.v. light using a novel design of disinfection unit.
METHODS Source and purification o f oocysts Oocysts of C. parvum were obtained from bovine faeces from a natural infection and were purified by sucrose and Percoll flotation. Purified oocysts were stored at 4~C in water.
2583
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Research Note
U.V. disinfection unit The u.v. disinfection unit is specifically designed for the purposes of inactivating Cryptosporidium and Giardia in clean water (Fig. 1). In brief, the unit consists of two treatment chambers each containing a 2/zm nominal filter. O n to each side of each filter are focused 3 LP Mercury A R C lamps (i.e. 6 lamps per filter), giving a theoretical m i n i m u m radiation intensity of approximately 14.58 m W cm 2, at the germicidal wavelength of 253.7 nm. The system is designed to capture any cysts or oocysts in the water on the first filter, then the flow within the filter chamber is reversed thereby back-flushing cysts or oocysts on to the second filter where they are trapped until a pre-set u.v. dose is reached. By the use of 8 valves, the pattern of water flow is designed to ensure that any cysts and oocysts will be captured on both membranes, but there will be no build up of debris within the system. The captured cysts or oocysts are thus exposed to a pre-set u.v. irradiation dose, totally independent of the flow rate. Flow rate through the system is designed to be approximately 2 0 0 m 3 h ~ when in use on-line. For these experiments the oocysts were trapped on each filter for a m a x i m u m of five minutes thereby exposing them to u.v. irradiation of approximately 8748 m W s cm 2, and the flow rate was set to be approximately 10 m 3 h ~. Experimental design Viability of oocysts was assessed by morphology and inclusion/exclusion of vital dyes (Campbell et al., 1992) before use and excysted according to Robertson et al. (1993). The excystation was examined after 30 min to ensure that motile sporozoites were observed. In experiments, conducted in triplicate, 5 × 107 oocysts in 2.5 ml water were added to the disinfection unit (equivalent to approximately I05 oocysts/l) and the u.v. mechanism switched on for 5 min on each of the two filters. Water in the unit was pre-filtered through a 1/~m yarn filter to minimise the presence of extraneous debris. For each run conducted with the u.v. mechanism switched on, a control experiment was run first in which the water and oocysts were pumped around the machine for 20 min but the u.v. mechanism was not switched on and valves were kept open to minimise flow through the machine's filters. Oocysts were then collected from the machine and their viability assessed. Temperature and pH were measured during each of the experiments.
Concentration o f oocysts from the disinfection unit To concentrate oocysts from the disinfection unit (total volume approx. 5001), a water sample (251) was drained from the purpose built side-arm of the machine sited immediately after the second filter/valve assembly. In the experimental runs, this was timed to capture a representative sample following the backflush of the oocysts from the filter. Samples obtained were then pumped through a 142 m m Sartorius filter (0.2/~m, nylon) and the filter cut up and washed in 200 ml 0.1% Tween-80 solution. This was then concentrated by a series of centrifugation steps at 1060g to give a final volume of 1 ml. Assessment o f ooeysts viability using the vital dye assay Viability of oocysts was assessed using the vital dye assay of Campbell et al. (1992) which relies upon morphology and inclusion/exclusion of two vital dyes, propidium iodide (PI) and 4'6 diamidino-2-phenyl indole (DAPI). A I h pre-incubation in acidified HBSS (Hanks' Balanced Salt Solution) (pH 2.75) was conducted prior to the incubation with the vital dyes in order to maximise vital dye uptake. In the final 30 min of incubation with the dyes, fluorescein isothiocyanate-labelled anti-Cryptosporidium monoclonal antibody was added to assist in the identification of the oocysts. At least 200 oocysts were assessed for viability in each trial and control.
Assessment o f viability using excystation Viability was also assessed by in vitro excystation of oocysts following the procedure of Robertson et al. (1993). Following a pre-incubation in acidified HBSS in order to maximise excystation, the oocysts were incubated in bile and sodium hydrogen carbonate in Hanks' Minimal Essential Medium. After 30 min and 4 h, aliquots of the excystation incubation were assessed by microscopy with enumeration of empty oocyst shells, non-excysted oocysts, partly excysted oocysts and motile and non-motile sporozoites. In trials 2 and 3 the aliquot taken after 30 min in excystation medium was examined in total by reading the whole sample contained beneath a 22 × 22 m m cover slip. Microscopy All microscopy was conducted on an Olympus BH-2 fluorescence microscope equipped with appropriate filter blocks for visualisation o f the dyes and with Nomarski
Fig. 1. Ultraviolet disinfection unit showing the two filtration/u.v, chambers.
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Table 1. Viability of control and experimental trial C. parvum oocysts by vital dye assay % non-viable Number of % viable (DAPI+/PI+ or % potentially oocysts (DAPI +/PI - ) empty shells) viable (DAPI -/PI - ) counted Control I 84 15 1 926 Control 2 86 14 -230 Control 3 89 11 -209 Trial 1 -100 -1164 Trial 2 -100 -216 Trial 3 -100 -211
optics for improved visualisation of the morphology for enumeration of the excystation.
DISCUSSION
Oocyst viability was considered to between 87 a n d 8 9 % before the trials were c o n d u c t e d with between 10 a n d I 1% o f the oocysts non-viable. The results o f the trials a n d controls are described in Tables 1-3. The viability o f the control oocysts was not significantly different to the initial viability before the oocysts were i n t r o d u c e d into the disinfection unit. Circulating the oocysts t h r o u g h the unit w i t h o u t the u.v. light a n d the subsequent c o n c e n t r a t i o n technique did not affect oocyst viability. D u r i n g the three trials the t e m p e r a t u r e of the water in the unit varied between 14-19°C a n d the p H varied between 7.9 a n d 8.2. A good correlation between the vital dye assay a n d the maximised 4 h excystation was observed; m e a n excystation in the control g r o u p was 9 0 % a n d the expected viability from the vital dye assay was 86%. Sporozoite ratio in the control samples was high after 30 min, but reduced after 4 h, p r e s u m a b l y due to lysis of sporozoites ( R o b e r t s o n et al. 1993). M o s t ( > 9 9 % ) free sporozoites observed in the excystation media were motile. Oocysts in the experimental samples also h a d their viability assessed by b o t h the vital dye assay a n d in vitro excystation. The vital dye assay indicated t h a t oocyst viability h a d been reduced by over 2logs, with no viable or potentially-viable oocysts observed. In vitro excystation confirmed the results of the vital dye assay, with no excystation observed. Free sporozoites were not observed after either 30 min or 4 h in the excystation media, even when the whole aliquot of the 3 0 m i n excystation (equivalent to approx. 10 s oocysts) b e n e a t h the coverslip was examined.
A l t h o u g h purification of water by u.v. t r e a t m e n t has been u n d e r consideration for several years, only in recent years has it been seriously considered as an option for potable water supplies (Snowball a n d Hornsey, 1988; Wolfe, 1990). However, the resistance of b o t h cysts of Giardia a n d the m o r e robust oocysts of C r y p t o s p o r i d i u m to u.v. systems tested has, to date, indicated t h a t u.v. t r e a t m e n t is unsuitable for treatm e n t of potable water supplies. These preliminary results indicate t h a t with the u.v. disinfection system used in this study there is the potential for inactivating oocysts o f C. p a r v u m in clean water. A l t h o u g h e n u m e r a t i o n of the experiment described here indicated between 2 a n d 3 l o g s reduction in viability of the oocysts (99.9% inactivation), it should be n o t e d that this figure is restricted by the limits of the s t a n d a r d e n u m e r a t i o n technique; extended e n u m e r a t i o n might be speculated to reveal the actual log reduction in viability to be greater t h a n this. The best t r e a t m e n t expected at present from a single procedure in water t r e a t m e n t is considered to be 3 log scale ( R o b e r t s o n et al. 1994), and these results indicate t h a t this disinfection system has the potential to match, or perhaps to exceed this figure. A multi-barrier a p p r o a c h has been considered the most practical a p p r o a c h to water t r e a t m e n t (Robertson et al. 1994) a n d this disinfection system, designed for treated water, m a y c o n t r i b u t e a valuable additional barrier. The t e m p e r a t u r e a n d p H at which these experiments were conducted would not be considered to have h a d any impact on the viability of the oocysts ( R o b e r t s o n et al. 1992; Fayer, 1994). Similar experiments have also been conducted o n the inactivation of a range of bacterial a n d viral cultures with a similar, a l t h o u g h not identical, u.v. disinfection unit (Snowball, M. R.; unpublished data). In these experiments, log kills have been
Table 2. Excystation of C. parvum oocysts in control and experimental trials after 30 mins in excystation medium Number of oocysts Excystation (%) Sporozoite ratio counted Control 1 66 3.8 174 Control 2 59 3.7 116 Control 3 30 4.2 154 Trial 1 --200 Trial 2 --200 Trial 3 --200
Table 3. Excystation of C. parvum oocysts in control and experimental trials after 4 hs in excystation medium Number of oocysts Excystation (%) Sporozoiteratio counted Control 1 89 0.2 208 Control 2 89 0.5 206 Control 3 92 0.4 209 Trial I --208 Trial 2 --218 Trial 3 --252
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Research Note
measured between 3 (Legionella spiritensis) to 7.2 (Salmonella typhimurium). These kills were considered to be in excess of those published for other u.v. disinfection systems with no p h o t o r e a c t i v a t i o n or resuscitation. CONCLUSIONS 1. These preliminary data suggest that the u.v. disinfection unit used for these experiments has the potential for inactivating oocysts of C. parvum in treated water, a n d thus m a y be of importance for inclusion "in-line" following conventional water treatment. 2. The 2-3 log reduction in oocyst viability suggested by the data presented in this note, is restricted by the limits o f the s t a n d a r d e n u m e r a t i o n technique; extended e n u m e r a t i o n by using a n autom a t e d c o u n t i n g system such as flow cytometry m i g h t be speculated to reveal the actual log reduction in viability to be greater t h a n this. 3. Whilst the a u t h o r s consider the data described in this c o m m u n i c a t i o n to be highly significant, it should be emphasised that the studies described represent only 3 trials with one isolate of oocysts and the following trials would also be r e c o m m e n d e d in order to verify these preliminary findings: (a) infectivity studies (b) experiments using different isolates of C. parvum, in particular with oocysts isolated from h u m a n infections, (c) studies with e n u m e r a t i o n of vital dye increased by several orders of m a g n i t u d e by using a n a u t o m a t e d c o u n t i n g system such as flow cytometry (d) Giardia trials: a l t h o u g h C. parvum oocysts are considered to be more r o b u s t t h a n Giardia cysts, it is i m p o r t a n t to test formally that Giardia cysts are also inactivated by this system. This is particularly pertinent as results from one study (Rice a n d Hoff, 1981) indicate that a u.v. dose of 63 m W s c m 2 resulted in between 7 0 - 8 0 % reduction in viability of Giardia cysts, while a n o t h e r group of workers ( R a n s o m e et al., 1993) report that the same u.v. dose applied in the same m a n n e r caused an 80% reduction in viability of C. parvum oocysts. A 9 0 % inactivation of Giardia muris cysts has also been reported (Carlson et al., 1990) at a u.v. dose o f 8 2 m W s c m 2. (e) Studies using a flow-through system in the o p e r a t i o n of the disinfection unit: in the experimental trials described here, the system used to facilitate sampling a n d c o n c e n t r a t i o n of oocysts required the water to be recirculated within the disinfection unit. This set-up restricted testing whether the 2 # m filter actually retained all the oocysts in a single pass. In order to ascertain that viable oocysts will not pass through the filter, a flow-through system should be assessed in further trials.
REFERENCES
Campbell A. T., Robertson L. J. and Smith H. V. (1992) Viability of Cryptosporidium parvum oocysts: correlation of in vitro excystation with inclusion/exclusion of fluorogenic vital dyes. Appl. environ. Microbiol. 58, 3488 3493. Carlson D. A. et al. (1985) Project summary: ultraviolet disinfection of water for small water supplies. Office of Research and Development, U.S. Environmental Protection Agency, Cincinnati, Ohio. EPA/600/S2-85/092. As quoted in Wolfe R. L. (1990). Ultraviolet disinfection of potable water: current technology and research needs. Environ. Svi. Technol 24, 768 773. DeLeon R., Rose J. B., Bosch A., Torrella F. and Gerba C. P. (1993) Detection of Giardia, Cryptosporidium and enteric viruses in surface and tap water samples in Spain. Int. J. environ. Hlth Res. 3, 121 129. Fayer R. (1994) Effect of high temperature on infectivity of Cryptosporidium parvum oocysts in water. Appl. environ. Microbiol. 60, 2732 2735. Korich D. G., Mead J. R., Madore M. S., Sinclair N. A, and Sterling C. R. (1990). Effects of ozone, chlorine dioxide, chlorine and monochloramine on Co,ptosporidium oocyst viability. Appl. environ. Microbiol. 56, 1423 1428. LeChevallier M. W., Norton W. D. and Lee R. G. (1991) Occurrence of Giardia and Cryptosporidium in surface water supplies. Appl. environ. Microbiol. 57, 2610-2616. Lorenzo-Lorenzo M. J., Ares-Mazas M. E., VillacortaMartinez de Maturana and Duran-Oreiro D. (1993) Effect of ultraviolet disinfection of drinking water on the viability of Cryptosporidium parvum oocysts. J. Parasitol. 79, 67-70. MacKenzie W. R., Hoxie N. J., Proctor M. E., Gradus M. S., Blair K. A., Peterson D. E., Kazmierczak J. J., Addiss D. G., Fox K. R., Rose J. B. and Davis J. P. (1994) A massive outbreak in Milwaukee of Crypto~poridium infection transmitted through the public water supply. N. Engl. J. Med. 331, 161 167. National Cryptosporidium Survey Group (1992) A survey of Cryptosporidium oocysts in surface and groundwaters in the UK. J. Inst. War. environ. Mgmt 6, 697-703. Ongerth J. E. and Stibbs H. H. (1987) Identification of Cryptay~oridium oocysts in river water. Appl. environ. Microbiol. 53, 672. Ransome M. E., Whitmore T. N. and Carrington E. G. (1993) Effect of disinfectants on the viability of Cryptosporidium parvum. Wat. Suppl. ll, Amsterdam, pp. 75-89. Rice E. W. and Hoff J. C. (1981) Inactivation of Giardia lamblia cysts by ultraviolet irradiation. Appl. environ. Microbiol. 42, 546 547. Robertson L. J., Campbell A. T. and Smith H. V. (1992) Survival of oocysts of Cryptosporidium parvum under various environmental pressures. Appl. environ. Microbiol. 58, 3494 3500. Robertson L. J., Campbell A. T. and Smith H. V. (1993) In vitro excystation of Cryptosporidiurn parvum. Parasitology 106, 13 19. Robertson L. J., Smith H. V. and Ongerth J. E. (1994) Cryptosporidium and cryptosporidiosis. Part III: Development of water treatment technologies to remove and inactivate oocysts. Microbiology Europe 2, 18 26. Smith H. V., Smith A. L., Girdwood R. W. A. and Carrington E. G. (1989) The effect of free chlorine on the viability of Cryptosporidium spp. oocysts. WRc, Bucks, U.K. Snowball M. R. and Hornsey I. S. (1988) Purification of water supplies using ultraviolet light. In Developments in Food Microbiology 3 (7) (Edited by Robinson R. K.), Vol. 3(7), pp. 171 191. Elsevier Applied Science. Wolfe R. L. (1990) Ultraviolet disinfection of potable water: current technology and research needs. Environ. Sci. Technol. 24, 768--773.