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Keywords:

  • drinking water disinfection;
  • Giardia lamblia;
  • medium-pressure UV;
  • Mongolian gerbils;
  • UV disinfection

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Aims:  In this study, we determined the ability of a promising alternative UV technology – a polychromatic emission from a medium-pressure UV (MP UV) technology – to inhibit the reactivation of UV-irradiated Giardia lamblia cysts.

Methods and Results:  A UV-collimated beam apparatus was used to expose shallow suspensions of purified G. lamblia cysts in PBS (pH 7·2) or filtered drinking water to a low dose (1 mJ cm−2) of MP UV irradiation. After UV irradiation, samples were exposed to two repair conditions (light or dark) and two temperature conditions (25°C or 37°C for 2–4 h). The inactivation of G. lamblia cysts by MP UV was very extensive, and c. 3 log10 inactivation was achieved with a dose of 1 mJ cm−2. Meanwhile, there was no apparent reactivation (neither in vivo nor in vitro) of UV-irradiated G. lamblia under the conditions tested.

Conclusion:  The results of this study indicated that, unlike the traditional low-pressure (LP) UV technology, an alternative UV technology (MP UV) could inhibit the reactivation of UV-irradiated G. lamblia cysts even when the cysts were exposed to low UV doses.

Significance and Impact of the Study:  It appears that alternative UV technology has some advantages over the traditional LP UV technology in drinking water disinfection because of their high level of inactivation against G. lamblia cysts and also effective inhibition of reactivation in UV-irradiated G. lamblia cysts.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Giardia lamblia (also known as Giardia duodenalis) is one of the most important waterborne pathogens in the world. It has been associated with numerous waterborne outbreaks over the last several decades (Marshall et al. 1997), many of which were associated with public drinking water supplies (Craun et al. 2003). G. lamblia cysts are ubiquitous in surface and source water (LeChevallier and Norton 1995) and resistant to conventional water treatment processes (Gibson et al. 1998). Although conventional filtration systems can achieve some removal of these cysts (Gibson et al. 1998), most chemical disinfection processes achieve only limited inactivation of G. lamblia cysts within practical doses and contact time (Sobsey 1989). Fortunately, it has been shown that traditional UV technology [low-pressure (LP) UV] is highly effective against G. lamblia cysts (Campbell and Wallis 2002; Linden et al. 2002; Mofidi et al. 2002). Furthermore, a recent study indicated that alternative UV technologies [medium-pressure (MP) UV and pulsed UV] are also remarkably effective against G. lamblia cysts, and a significant inactivation (e.g. 4 log10) of G. lamblia cysts can be achieved at very low UV doses (Shin et al. 2009).

When it comes to UV irradiation, however, the ability of UV-irradiated micro-organisms to repair their UV damage should be addressed. It is well known that many waterborne bacteria such as many indicator bacteria (Whitby et al. 1984; Harris et al. 1987) and some pathogenic bacteria (Das et al. 1981; The United States Environmental Protection Agency (US EPA) 1986) do have one or more of repair pathways and repair DNA lesions caused by UV irradiation. Although there are two studies on the reactivation of G. lamblia after exposure to traditional LP UV technology (Linden et al. 2002; Li et al. 2008), nothing is known about the reactivation of G. lamblia cysts after exposure to alternative UV technologies such as MP UV. Interestingly, it was reported that Giardia muris cysts exposed to an alternative UV technology (MP UV) showed an ability to restore infectivity under certain repair conditions (Belosevic et al. 2001). However, G. muris is a murine pathogen and there might be significant physiological differences between G. muris and G. lamblia (a human pathogen) that may result in different responses to alternative UV technologies. Therefore, the primary objective of this study was to determine the presence and the extent of reactivation of G. lamblia cysts after exposures to an alternative UV technology (MP UV).

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Giardia lamblia cysts

Giardia lamblia cysts were purchased from Parasitology Research Labs, Neosho, MO, USA. Shed cysts collected from experimentally infected Mongolian gerbils were screened to remove large debris, then mixed with zinc sulfate solution (ZnSO4, 1·2 specific gravity), and centrifuged at 300 g for 5 min. Cysts recovered from supernatant were washed with distilled water, resuspended in buffer solution containing antibiotics, and stored at 4°C.

UV disinfection

MP UV irradiation system and radiometry.  The bench-scale, MP UV–collimated beam apparatus consisted of a 400- W MP UV lamp (Model 7825 Immersion Lamp; Hanovia Ltd., Slough, UK) mounted on a housing above a quasi-collimating cylinder. Germicidal UV irradiance emitted from the broadband MP UV lamp was measured with the International Light IL1700 radiometer (International Light Inc., Newburyport, MA, USA). The dose was weighted by the DNA absorbance spectrum according to a spreadsheet developed by Dr James Bolton (Bolton 2002). The delivered UV dose, accounting for the depth of the suspension (0·255 cm), was calculated based on the measurement of the irradiance incident on the petri dish, a series of correction factors (petri factor, reflection factor, water factor, divergence factor, sensor factor, and germicidal factor) as described in Bolton and Linden (Bolton and Linden 2003), and the exposure time in seconds.

Experimental protocol of UV disinfection experiments. Giardia lamblia cysts were mixed and diluted in PBS (pH 7·2) or a filtered drinking water (Orange County Water and Sewer Authority, Chapel Hill, NC, USA) to give final concentrations of c. 105 cysts per ml. Small aliquots (5 ml) in 60 × 15 mm cell culture petri dishes were irradiated with the aforementioned collimated beam type UV source while stirring the samples slowly on a magnetic stir plate. After predetermined exposure times, samples were removed from the UV irradiation systems and were subjected to reactivation experiments.

Reactivation experiments

To verify our reactivation experiment protocol, reactivation experiments were first performed with Escherichia coli B that is known to have the ability to repair their UV-damaged DNA (Setlow and Carrier 1964). Escherichia coli B was acquired from available stocks of the Environmental Microbiology Laboratory at the University of North Carolina at Chapel Hill. It was found that our protocol has the ability to facilitate an appreciable reactivation of E. coli B under both light and dark repair conditions (for example, we observed c. 1 log10 reactivation of E. coli B when it was first irradiated with 7 mJ cm−2 (that resulted in 4 log10 initial inactivation of E. coli B) and then exposed to the light repair condition at 25°C). For reactivation experiments with G. lamblia cysts, samples with c. 105G. lamblia cysts in 5 ml of PBS (pH 7·2) in 60 × 15 mm cell culture petri dishes were first exposed to a low dose (1 mJ cm−2) of UV irradiation and then wrapped with aluminum foil immediately after UV exposure. One sample was kept at 4°C as an experimental control. Duplicate samples were transferred to 25 or 37°C incubators. One dish at each temperature was illuminated by an 8 W fluorescent lamp (F8T5WW; GE, Nela Park, Cleveland, OH, USA) at a distance of c. 15 cm with slow stirring (light repair), and the other was stirred while kept wrapped with aluminum foil (dark repair). After incubation (2–4 h), the samples were immediately serially diluted 10-fold in PBS (pH 7·2) and inoculated into animals for infectivity assay.

Gerbil infectivity assay

Giardia lamblia infectivity assays were performed in 8- to 10-week-old female Mongolian gerbils (Meriones unguiculatus) as previously described (Belosevic et al. 1983). Briefly, the gerbils were purchased from Charles River, Canada (St Constant, QC, Canada). At least 10 days before experimental infection, the animals were treated once with a solution (20 mg per gerbil) of metrodinazole (Flagyl; Rhone Poulenc, Montreal, QC, Canada) which was administered by gavage. This treatment ensured that the gerbils were free from all previous intestinal infections (including Giardia), as demonstrated by three consecutive examinations of faeces. Faeces were collected daily starting 3 days after infection and lasting until day 25. This period of collection was chosen because it represents the latent, the acute, and the elimination phase of the Giardia infection in gerbils (Belosevic et al. 1983). The presence of cysts released in a 2-h faecal collection (10 am to noon) by individual gerbils was determined by the sucrose flotation method as described previously (Belosevic and Faubert 1983).

Data presentation

The infectivity titres of G. lamblia cysts were calculated as a most probable number (MPN) based on the presence/absence of G. lamblia cysts in individual gerbils inoculated with a specified number of cysts (Table 1). First, the outcome of infectivity assay was recorded as ‘infectivity response’ that is the number of gerbils passing cysts in their stool divided by total number of gerbils infected with the specific number of cysts (‘cyst dose’). Then, the ‘infectivity MPN’ of a specific sample was calculated based on the ‘infectivity response’s of all the dilutions (‘cyst dose’) of the specific sample. (An example of how to calculate ‘infectivity MPN’ is given in the footnotes of Table 1.) Finally, the MPN log10 reduction was calculated by dividing the MPN of a specific sample by the initial MPN of the control (unexposed) sample and taking the base 10 log of that result.

Table 1.   Reactivation of UV-irradiated Giardia lamblia cysts in PBS (pH 7·2) after exposure to a dose of 1 mJ cm−2 of MP UV and four different repair conditions
UV dose (mJ cm−2)Cyst dose*Infectivity response†Cyst doseInfectivity responseCyst doseInfectivity responseInfectivity MPNLog reduction (log10Nd/No)
  1. MP, medium pressure; MPN, most probable number.

  2. *Cyst dose is the number of G. lamblia cysts dosed into each animal.

  3. †Infectivity response is the number of gerbils passing cysts in the stool/total number of animals infected.

  4. ‡Repair conditions: 25/L = 25°C, light repair, 2 h, 25/D = 25°C, dark repair, 4 h, 37/L = 37°C, light repair, 2 h, and 37/D = 37°C, dark repair, 4 h.

  5. §An example of how to calculate an ‘infectivity MPN’: For the data set in the Row 2:1, where three of four gerbils became infected when each gerbil was inoculated with 1000 cysts and three of four gerbils became infected when each gerbil was inoculated with 10 000 cysts, the MPN/million cysts = (No. of positive animals × 1 000 000)/((number of cysts in negative animals) × (number of cysts in all animals))½ = ((3 + 3) × 1 000 000)/(((1000 × 1) + (10000 × 1)) × ((1000 × 4) + (10000 × 4)))½ = 273 infectious cysts/million total cysts).

0, control104/41004/4  >121 000 
1  10003/4100003/4273§>2·65
25/L‡100/41003/410002/41620>1·87
25/D  1000/410000/4227>2·73
37/L100/41000/410000/4226>2·73
37/D100/41000/410000/4226>2·73

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Table 1 summarizes the results of reactivation experiments for G. lamblia cysts in PBS (pH 7·2) after exposure to a dose of 1 mJ cm−2 of MP UV and then to four different repair conditions [light repair at 25°C (25L), dark repair at 25°C (25D), light repair at 37°C (37L), and dark repair at 37°C (37D)]. The raw gerbil infectivity assay data, the computed MPN, and the MPN log10 reductions are reported in Table 1. The infectivity of G. lamblia cysts was very high, and all the four gerbils were infected with only 10 cysts (Row 1: ‘0, control’). Inactivation of G. lamblia cysts by MP UV was fast and reached the detection limit of the gerbil infectivity assay (2·65 log10 for this experiment) with a UV dose of 1 mJ cm−2 (Row 2: ‘1’). Meanwhile, there was no apparent restoration of G. lamblia cyst infectivity following exposure of the UV-irradiated cysts to either light or dark repair conditions (Row 3–6: ‘25L, 25D, 37L, 37D’) except a slight increase in the infectivity in one condition (Row 3: ‘25L’).

Table 2 shows the results of reactivation experiments for G. lamblia cysts in a filtered drinking water after exposure to a dose of 1 mJ cm−2 of MP UV and to the four repair conditions. As in the buffered water (PBS), the inactivation of G. lamblia cysts by MP UV was fast and reached the detection limit of the gerbil infectivity assay (3·74 log10 for this experiment) within a UV dose of 1 mJ cm−2 (Row 2: ‘1’). Again, there was no apparent restoration of G. lamblia cyst infectivity following exposure of the UV-irradiated cysts to either light or dark repair conditions (Row 3–6: ‘25L, 25D, 37L, 37D’) except a slight increase in the infectivity in one condition (Row 3: ‘25L’).

Table 2.   Reactivation of UV-irradiated Giardia lamblia cysts in a filtered drinking water after exposure to a dose of 1 mJ cm−2 of MP UV and four different repair conditions
UV dose (mJ cm−2)Cyst dose*Infectivity response†Cyst doseInfectivity responseCyst doseInfectivity responseInfectivity MPNLog reduction (log10Nd/No)
  1. MP, medium pressure; MPN, most probable number.

  2. *†‡The same as the footnotes in Table 1.

0, control54/4504/4  >240 000 
1  50002/4275222/444>3·74
25/L‡503/45003/450003/4810>2·47
25/D  5002/450000/493>3·41
37/L500/45000/450000/4<45>3·73
37/D500/45001/450001/452>3·66

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

The results of this study indicate that there is no evidence of in vivo reactivation of G. lamblia cysts after exposure to an alternative UV technology (MP UV). As mentioned in the Materials and methods, the signs of infection by UV-irradiated G. lamblia cysts in gerbils were observed for 25 days. Therefore, it appears that there is no in vivo reactivation of UV-irradiated G. lamblia cysts in gerbils unless there is an unusually long lag period in reactivation. This result is in contrast to the finding of a previous study reporting in vivo reactivation of G. muris cysts after exposure to MP UV irradiation (Belosevic et al. 2001). It is not certain if this discrepancy between the two studies is because of the difference in the species of Giardia (lamblia vs muris), the experimental animals (gerbils vs mice), the UV doses used (1 vs 25 mJ cm−2), the suspension waters used, or other experimental factors.

Also, the results of this study suggest that there is no significant in vitro reactivation of G. lamblia cysts after exposure to MP UV under the reactivation conditions tested. The reactivation conditions in this study were the ones commonly used in UV reactivation studies in bacterial and mammalian cells (Das et al. 1981; Harris et al. 1987), and it was found that our reactivation protocol has the ability to facilitate an appreciable reactivation of E. coli B under both light and dark repair conditions. However, there was no apparent restoration of G. lamblia cysts infectivity in this study following exposure of UV-irradiated G. lamblia cysts to the four reactivation conditions. Nonetheless, it is still possible that UV-irradiated G. lamblia cysts might reactivate in different conditions such as longer incubation time and/or presence of some environmental stimulants that would turn UV-irradiated G. lamblia cysts into a metabolically active state (trophozoites).

Finally, it appears that different UV technologies have different ability in terms of preventing repair activity in G. lamblia cysts. In fact, previous studies reported that there were different levels of repair activity in E. coli when E. coli was irradiated with different UV technologies (LP and MP UV) (Oguma et al. 2002; Zimmer and Slawson 2002). Although our previous study (Linden et al. 2002) showed that there was no reactivation of G. lamblia cysts when they were exposed to high doses (16 and 40 mJ cm−2) of traditional UV technology (LP UV), a recent study (Li et al. 2008) suggested that G. lamblia trophozoites reactivated when they were exposed to low doses (up to 10 mJ cm−2) of LP UV. Although trophozoites are not the environmental form of G. lamblia that are subjected to UV disinfection in water and wastewater treatment processes, it is still possible that UV-irradiated G. lamblia cysts were exposed to certain conditions to make them excyst and turned into metabolically active trophozoites. In fact, the reactivation conditions used in this study were the ones potentially to stimulate the UV-irradiated G. lamblia cysts into a metabolically active state. However, there is no apparent reactivation of G. lamblia after exposure to a low dose (1 mJ cm−2) of MP UV technology in this study. Overall, it appears that alternative UV technology (MP UV) can provide an adequate reduction in G. lamblia cysts in drinking water because of its high level of inactivation against G. lamblia cysts and also effective inhibition of reactivation in UV-irradiated G. lamblia cysts, which would give this alternative UV technology some advantages over the traditional LP UV technology in drinking water disinfection.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

This research was supported by funds from the National Science Foundation (BES-0533529).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References
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