- Email: [email protected]
Low- and medium-pressure UV inactivation of microsporidia Encephalitozoon intestinalis
Water Research 36 (2002) 3161–3164
Research note
Low- and medium-pressure UV inactivation of microsporidia Encephalitozoon intestinalis Debra E. Huffmana,*, Angela Gennaccaroa, Joan B. Rosea, Bertrand W. Dussertb a
College of Marine Science, University of South Florida, 140 7th Ave. South, St. Petersburg, FL 33701, USA b USFilter Wallace and Tiernan Products, 1901 West Garden Road, Vineland, NJ 08360, USA Received 1 September 2001; accepted 1 November 2001
Abstract Newly recognized waterborne pathogens such as microsporidia are being detected in the world’s water supplies with increasing frequency. Many of these organisms have been shown to cause negative health impacts for both immunocompetent as well as immunocompromised individuals. It is imperative that these emerging pathogens be investigated for their ability to resist both traditional and novel disinfection technologies that are currently in use or under consideration for drinking water treatment. Low- and medium pressure UV light is at the cutting edge of disinfection technologies for the drinking water industry. While previous UV disinfection studies have focused on the inactivation of Cryptosporidium and Giardia as well as viruses and common bacteria, this research reports the ability of low- and medium pressure UV light to inactivate >3.6 log10 of microsporidia Encephalitozoon intestinalis spores at a dose of 6 mJ/cm2 or higher as determined using a cell culture approach. Published by Elsevier Science Ltd. Keywords: UV light; Disinfection; Microsporidia; Cell culture
1. Introduction Microsporidia are obligate intracellular spore-forming protozoan parasites that are capable of infecting both vertebrate and invertebrate hosts. Their role as an emerging pathogen in immunocompetent as well as immunosuppressed hosts is being increasingly recognized. The prevalence of microsporidiosis in studies of AIDS patients with chronic diarrhea ranges from 7% to 50% worldwide [1]. It is unclear whether this broad range represents geographic variation, differences in diagnostic capabilities or differences in risk factors for exposure to microsporidia. While AIDS patients account for the largest portion of the immunocompromised population, immune suppression has become a common phenomenon in modern medicine for the *Corresponding author. Tel.: +1-727-553-3946; fax: +1727-553-1189. E-mail address: [email protected] (D.E. Huffman). 0043-1354/02/$ - see front matter Published by Elsevier Science Ltd. PII: S 0 0 4 3 - 1 3 5 4 ( 0 1 ) 0 0 5 2 8 - 0
treatment of severe inflammatory diseases, during chemotherapy, during treatment of allergies and after organ transplantation. This ever-increasing immunocompromised portion of the world’s population is at increased risk of infection from emerging pathogens such as microsporidia. In the immunocompetent individual, microsporidia may be asymptomatic or have a self-limiting diarrheal illness [1,2]. Typical symptoms of infection with microsporidia are similar to those noted for other protozoan parasites and include chronic diarrhea, dehydration and significant weight loss (>10% body weight) [2]. A single reported drinking water outbreak of microsporidiosis occurred in the summer of 1995 in France [2]. Approximately 200 cases of microsporidiosis were identified, mostly in AIDS patients. The major factor associated with diagnosis was living in an area corresponding to one of the three water distribution subsystems in the town. Contamination of the drinking water with untreated lake water was suspected. The small size of this organism (1–5 mm) makes it difficult to remove using
3162
D.E. Huffman et al. / Water Research 36 (2002) 3161–3164
conventional filtration techniques found in many drinking water treatment plants [2]. This organism has also been shown to be extremely stable in the environment with several species remaining infective for months to years outside their hosts [3–5]. Therefore, the use of chemical or physical disinfectants for drinking water treatment is critical in the prevention of future waterborne outbreaks of microsporidiosis.
2. Materials and methods 2.1. UV apparatus All irradiation experiments were carried out using a Rayoxs bench-scale collimated beam unit equipped with either a proprietary 1 kW medium-pressure UV lamp or a 10 W low-pressure UV lamp mounted in a housing above a polyvinyl chloride cylinder to collimate the beam (Model PS1-1-120, Calgon Carbon Corporation). This apparatus produces a nearly parallel beam of UV radiation by means of a 6 cm 93 cm long collimating tube placed below the UV lamp. A plastic petri dish (60 mm diameter 15 mm height) was placed on a stir plate that was centered 15 cm beneath the open end of the collimating tube. The E. intestinalis spores, obtained from the American Type Culture Collection (ATCC # 50506, Rockville, MD), were suspended in 150 mM phosphate buffered saline (PBS) which was prepared by dissolving appropriate amounts of reagent grade potassium dihydrogen sodium phosphate and disodium hydrogen phosphate (Fisher Scientific, Atlanta, GA) in deionized milliQ water. The final PBS solution was adjusted to pH 7.2. The test volume of PBS was 15 mL and the liquid height in the petri dish was 8.5 mm. Suspensions were gently and constantly stirred during the exposure period by a 10 mm 3 mm magnetic stir bar. No heat was produced by the mixer. All testing was performed under room temperature conditions (231C). The duration of the exposure was controlled by means of a pneumatic shutter located at the top of the collimating tube, beneath the UV lamp and was measured with a stopwatch. Process controls were similarly manipulated but were not exposed to UV light. 2.2. UV dose calculations The UV irradiance at the surface of the liquid and at the center of the UV beam was measured immediately before and after each experiment. The measurement was made with an International Light Model IL1400A radiometer equipped with a SED240 UV detector calibrated by the manufacturer to standards of the National Institute of Standards and Technology (NIST). The percent transmitance of the E. intestinalis spore
suspensions (1 cm cell at 254 nm) ranged from 88.0% to 96.6% depending on the purity of the spore stock suspension. The effective average irradiance was determined as described by Craik et al. [6]. 2.3. In-vitro cell culture A rabbit kidney cell line (RK-13) ATCC #CCL-37, was utilized to produce the E. intestinalis spores as well as to evaluate the disinfection effectiveness of both lowand medium-pressure UV exposure. The RK-13 cells were maintained in 75 cm2 tissue culture flasks in a 5% CO2 environment at 371C. Cells were passaged weekly by trypsinization with one part PBS-EDTA and one part 0.25% trypsin. 2.4. E. intestinalis spore production E. intestinalis spores were reconstituted with sterile cell culture maintenance media and inoculated into 75 cm2 flasks containing RK-13 cells. Maintenance media for cell growth consisted of 20 mLs of Eagles Minimal Essential Media (ATCC, Rockville, MD) supplemented with 5% fetal bovine serum (Atlanta Biologicals, Norcross, GA), 03% hepes buffer (pH 7.3; Sigma, St. Louis, MO), 1% 200 mM L-glutamine (Sigma, St. Louis, MO). The media was prepared as described with an increase in the concentration of fetal bovine serum (FBS) to 8% (growth media) when the RK-13 cells were infected with E. intestinalis spores. Media in the infected flasks were concentrated by centrifugation (1500g for 5 min) and added back to the flasks along with fresh growth media on a weekly basis. Infected flasks were also supplemented when necessary with freshly trypsinized cells from uninfected flasks until the production of high concentration E. intestinalis spores, as determined by hemacytometer, could be detected in the cell culture media. No antibiotics were used during any of the cell culture procedures. Production of high titer spores required approximately 3 to 4 weeks of growth in culture. After this point in time, spores were harvested from the media and purified using percol density gradients. After final concentration, the spores were resuspended in PBS and enumerated in triplicate using a Neubauer hemacytometer. The spore concentration was adjusted such that a concentration of approximately 1 105 spores/mL in 50 mL of PBS was obtained. Purified spores of E. intestinalis were stored at 41C and were o2 weeks of age when evaluated for their resistance to UV disinfection. 2.5. Sample collection post UV treatment E. intestinalis samples of 1 mL were collected directly from the petri dish following UV exposure using an eppendorf pipet equipped with a disposable aerosol
D.E. Huffman et al. / Water Research 36 (2002) 3161–3164
3163
barrier pipet tip. The samples were placed in 2 mL eppendorf tubes from which 10-fold dilutions were prepared using RK-13 growth media. Samples were diluted from initial spore concentrations of 105 to 100/ mL in 900 mL of growth media. Each of the dilutions was then pipetted in 150 ml aliquots onto 6 replicate wells of 2 day old RK-13 monolayers grown in Lab Tech-II chamber slides (Nalgene Nunc, Naperville, Ill) that showed 85–90% confluent cell growth. The two uninfected wells on each slide served as negative controls. Sample processing and cell culture inoculation occurred within 1 h of UV exposure. 2.6. Cell culture analysis UV treated samples and controls were analyzed using a modified foci detection method-most probable number (FDM-MPN) cell culture technique [7,8]. Modifications included extending the incubation period from 2 days post infection (as performed for Cryptosporidium) to 5 days for microsporidia. On the fifth day post infection, the well chamber slides were removed from the incubator, the media aspirated and the upper chamber portion removed. The slides were then fixed for 10 min in 100% methanol (Sigma, St. Louis, MO) and allowed to air dry. The slides were then stained in 0.05% solution of Calcofluor White M2R in 0.1 M phosphate buffered saline (pH 7.0–8.0) (Sigma, St. Louis, MO) for 15 s and counter stained in 0.2% Evans Blue for 5 s [7]. After the slides were dry, cover slips were attached using nail polish and the slides were placed in the dark at room temperature until microscopic evaluation was performed. Stained monolayers were evaluated using epifluorescence microscopy (excitation filter 340–380 nm) at 200 and confirmed at 400 magnification. Each well on the chamber slide was viewed and scored as either positive (infection present) or negative (no infection). When infection was present, large clusters of infectious E. intestinalis spores were easily identified against the dark blue background of uninfected RK-13 cells. The MPN of infectious spores was determined using the Information Collection Rule (ICR) Most Probable Number calculator, version 1.00 [9].
3. Results and discussion Replicate cell culture analyses were performed using three UV doses for both low- and medium-pressure UV (Fig. 1). The control spores of E. intestinalis had a mean percent infectivity of 2.1%70.3% with an average MPN/mL of 8.1 103 and a standard deviation of 2.1 103 based upon an average initial concentration of 3.8 105/mL74.0 104 spores/mL. The spores that were exposed to either low- or medium-pressure UV
Fig. 1. Low- and medium-pressure UV inactivation of Microsporidia E. intestinalis. Error bars indicate 95% confidence intervals.
doses of 6 and 9 mJ/cm2 showed no infectious foci in any of the cell monolayers yielding >3.9–>4.0 log10 inactivation of E. intestinalis. A low- or medium-pressure dose of 3 mJ/cm2 showed an inactivation of 1.6–2.0 log10. Previous studies using the FDM-MPN to evaluate the effectiveness of low- and medium-pressure UV for the inactivation of Cryptosporidium parvum showed slightly higher oocyst inactivation at a dose of 3 mJ/cm2 (2.0– 2.9 log10) [10]. Previous studies of low- and mediumpressure UV light inactivation of C. parvum using mouse infectivity analysis have shown tailing in the UV inactivation curve at doses >25 mJ/cm2 [6]. This tailing was not noted in previous FDM-MPN assays for Cryptosporidium post UV exposure [10]. Replicate experiments showed minimal change in the infectivity of the stock culture of E. intestinalis throughout the time frame of the study. All of the microsporidia stocks were o30 days of age at the time of assay and the TCID50, infectious dose at which 50% of the tissue culture wells were positive, was 55 spores. Previous dose response data for E. intestinali in a cell culture system reported a TCID50 of 915 spores [11]. The potential use of UV disinfection for the inactivation of protozoa in drinking water has been steadily growing since it was determined that both medium and low-pressure UV could inactivate Cryptosporidium oocysts and Giardia cysts at doses that are easily achievable for drinking water treatment. Microsporidia has been placed on the EPAs candidate contaminate list as potentially requiring regulation and monitoring. This research demonstrates that UV light at dosages utilized for drinking water treatment is capable of achieving high levels of inactivation of microsporidia E. intestinalis.
Acknowledgements This work was supported by Calgon Carbon Corporation, Pittsburgh, PA. At the time of the research,
3164
D.E. Huffman et al. / Water Research 36 (2002) 3161–3164
Dr. B. Dussert was the Director of UV Research at Calgon Carbon Corp. [7]
References [1] Bryan RT. Microsporidiosis as an AIDS-related opportunistic infection. Clin Infect Dis 1995;21(1):62–5. [2] Cotte L, Rabodonirina M, Chapuis F, Bailly F, Bissuel F, Raynal C, Gelas P, Persat F, Piens M-A, Trepo C. Waterborne outbreak of intestinal microsporidiosis in persons with and without human immunodeficiency virus infection. J Infect Dis 1999;180:2003–8. [3] Shadduck JA, Polley MB. Some factors influencing the invitro infectivity and replication of Encephalitozoon cuniculi. J Protozool 1978;25:491–6. [4] Shadduck JA. Human microsporidiosis and AIDS. Rev Infect Dis 1989;11:203–7. [5] Waller T. Sensitivity of Encephalitozoon cuniculi to various temperatures disinfectants and drugs. Lab Anim 1979;13:227–30. [6] Craik SA, Weldon D, Finch GR, Bolton JR, Belosevic M. Inactivation of Cryptosporidium parvum oocysts using
[8]
[9]
[10]
[11]
medium- and low-pressure ultraviolet radiation. Water Res 2001;35(6):1387–98. John D. Masters thesis: Effects of Chemical Disinfection on Microsporidia E. Intestinalis. University of Arizona Department of Soil and Water Science, Tucson, Arizona, 2000. Slifko TR, Huffman DE, Rose JB. A most-probable number assay for enumeration of infectious Cryptosporidium parvum oocysts. Appl Environ Microbiol 1999;65(9):3936–41. Klee AJ. ICR most probable number calculator, Version 1.00 [online], Risk Reduction Laboratory, US Environmental Protection Agency, Cincinnati, OH, 1999. http:// www.epa.gov/nerlcwww/other.htm Huffman DE. Unpublished data. University of South Florida, College of Marine Science 140 7th Ave. South, St. Petersburg, FL 33701, 2001. Wolk DM, Johnson CH, Rice EW, Marshall MM, Grahn KF, Plummer CB, Sterling CR. A spore counting method and cell culture model for chlorine disinfection studies of Encephalitozoon syn. Septata intestinalis. Appl Environ Microbiol 2000;66(4):1266–73.
Research note
Low- and medium-pressure UV inactivation of microsporidia Encephalitozoon intestinalis Debra E. Huffmana,*, Angela Gennaccaroa, Joan B. Rosea, Bertrand W. Dussertb a
College of Marine Science, University of South Florida, 140 7th Ave. South, St. Petersburg, FL 33701, USA b USFilter Wallace and Tiernan Products, 1901 West Garden Road, Vineland, NJ 08360, USA Received 1 September 2001; accepted 1 November 2001
Abstract Newly recognized waterborne pathogens such as microsporidia are being detected in the world’s water supplies with increasing frequency. Many of these organisms have been shown to cause negative health impacts for both immunocompetent as well as immunocompromised individuals. It is imperative that these emerging pathogens be investigated for their ability to resist both traditional and novel disinfection technologies that are currently in use or under consideration for drinking water treatment. Low- and medium pressure UV light is at the cutting edge of disinfection technologies for the drinking water industry. While previous UV disinfection studies have focused on the inactivation of Cryptosporidium and Giardia as well as viruses and common bacteria, this research reports the ability of low- and medium pressure UV light to inactivate >3.6 log10 of microsporidia Encephalitozoon intestinalis spores at a dose of 6 mJ/cm2 or higher as determined using a cell culture approach. Published by Elsevier Science Ltd. Keywords: UV light; Disinfection; Microsporidia; Cell culture
1. Introduction Microsporidia are obligate intracellular spore-forming protozoan parasites that are capable of infecting both vertebrate and invertebrate hosts. Their role as an emerging pathogen in immunocompetent as well as immunosuppressed hosts is being increasingly recognized. The prevalence of microsporidiosis in studies of AIDS patients with chronic diarrhea ranges from 7% to 50% worldwide [1]. It is unclear whether this broad range represents geographic variation, differences in diagnostic capabilities or differences in risk factors for exposure to microsporidia. While AIDS patients account for the largest portion of the immunocompromised population, immune suppression has become a common phenomenon in modern medicine for the *Corresponding author. Tel.: +1-727-553-3946; fax: +1727-553-1189. E-mail address: [email protected] (D.E. Huffman). 0043-1354/02/$ - see front matter Published by Elsevier Science Ltd. PII: S 0 0 4 3 - 1 3 5 4 ( 0 1 ) 0 0 5 2 8 - 0
treatment of severe inflammatory diseases, during chemotherapy, during treatment of allergies and after organ transplantation. This ever-increasing immunocompromised portion of the world’s population is at increased risk of infection from emerging pathogens such as microsporidia. In the immunocompetent individual, microsporidia may be asymptomatic or have a self-limiting diarrheal illness [1,2]. Typical symptoms of infection with microsporidia are similar to those noted for other protozoan parasites and include chronic diarrhea, dehydration and significant weight loss (>10% body weight) [2]. A single reported drinking water outbreak of microsporidiosis occurred in the summer of 1995 in France [2]. Approximately 200 cases of microsporidiosis were identified, mostly in AIDS patients. The major factor associated with diagnosis was living in an area corresponding to one of the three water distribution subsystems in the town. Contamination of the drinking water with untreated lake water was suspected. The small size of this organism (1–5 mm) makes it difficult to remove using
3162
D.E. Huffman et al. / Water Research 36 (2002) 3161–3164
conventional filtration techniques found in many drinking water treatment plants [2]. This organism has also been shown to be extremely stable in the environment with several species remaining infective for months to years outside their hosts [3–5]. Therefore, the use of chemical or physical disinfectants for drinking water treatment is critical in the prevention of future waterborne outbreaks of microsporidiosis.
2. Materials and methods 2.1. UV apparatus All irradiation experiments were carried out using a Rayoxs bench-scale collimated beam unit equipped with either a proprietary 1 kW medium-pressure UV lamp or a 10 W low-pressure UV lamp mounted in a housing above a polyvinyl chloride cylinder to collimate the beam (Model PS1-1-120, Calgon Carbon Corporation). This apparatus produces a nearly parallel beam of UV radiation by means of a 6 cm 93 cm long collimating tube placed below the UV lamp. A plastic petri dish (60 mm diameter 15 mm height) was placed on a stir plate that was centered 15 cm beneath the open end of the collimating tube. The E. intestinalis spores, obtained from the American Type Culture Collection (ATCC # 50506, Rockville, MD), were suspended in 150 mM phosphate buffered saline (PBS) which was prepared by dissolving appropriate amounts of reagent grade potassium dihydrogen sodium phosphate and disodium hydrogen phosphate (Fisher Scientific, Atlanta, GA) in deionized milliQ water. The final PBS solution was adjusted to pH 7.2. The test volume of PBS was 15 mL and the liquid height in the petri dish was 8.5 mm. Suspensions were gently and constantly stirred during the exposure period by a 10 mm 3 mm magnetic stir bar. No heat was produced by the mixer. All testing was performed under room temperature conditions (231C). The duration of the exposure was controlled by means of a pneumatic shutter located at the top of the collimating tube, beneath the UV lamp and was measured with a stopwatch. Process controls were similarly manipulated but were not exposed to UV light. 2.2. UV dose calculations The UV irradiance at the surface of the liquid and at the center of the UV beam was measured immediately before and after each experiment. The measurement was made with an International Light Model IL1400A radiometer equipped with a SED240 UV detector calibrated by the manufacturer to standards of the National Institute of Standards and Technology (NIST). The percent transmitance of the E. intestinalis spore
suspensions (1 cm cell at 254 nm) ranged from 88.0% to 96.6% depending on the purity of the spore stock suspension. The effective average irradiance was determined as described by Craik et al. [6]. 2.3. In-vitro cell culture A rabbit kidney cell line (RK-13) ATCC #CCL-37, was utilized to produce the E. intestinalis spores as well as to evaluate the disinfection effectiveness of both lowand medium-pressure UV exposure. The RK-13 cells were maintained in 75 cm2 tissue culture flasks in a 5% CO2 environment at 371C. Cells were passaged weekly by trypsinization with one part PBS-EDTA and one part 0.25% trypsin. 2.4. E. intestinalis spore production E. intestinalis spores were reconstituted with sterile cell culture maintenance media and inoculated into 75 cm2 flasks containing RK-13 cells. Maintenance media for cell growth consisted of 20 mLs of Eagles Minimal Essential Media (ATCC, Rockville, MD) supplemented with 5% fetal bovine serum (Atlanta Biologicals, Norcross, GA), 03% hepes buffer (pH 7.3; Sigma, St. Louis, MO), 1% 200 mM L-glutamine (Sigma, St. Louis, MO). The media was prepared as described with an increase in the concentration of fetal bovine serum (FBS) to 8% (growth media) when the RK-13 cells were infected with E. intestinalis spores. Media in the infected flasks were concentrated by centrifugation (1500g for 5 min) and added back to the flasks along with fresh growth media on a weekly basis. Infected flasks were also supplemented when necessary with freshly trypsinized cells from uninfected flasks until the production of high concentration E. intestinalis spores, as determined by hemacytometer, could be detected in the cell culture media. No antibiotics were used during any of the cell culture procedures. Production of high titer spores required approximately 3 to 4 weeks of growth in culture. After this point in time, spores were harvested from the media and purified using percol density gradients. After final concentration, the spores were resuspended in PBS and enumerated in triplicate using a Neubauer hemacytometer. The spore concentration was adjusted such that a concentration of approximately 1 105 spores/mL in 50 mL of PBS was obtained. Purified spores of E. intestinalis were stored at 41C and were o2 weeks of age when evaluated for their resistance to UV disinfection. 2.5. Sample collection post UV treatment E. intestinalis samples of 1 mL were collected directly from the petri dish following UV exposure using an eppendorf pipet equipped with a disposable aerosol
D.E. Huffman et al. / Water Research 36 (2002) 3161–3164
3163
barrier pipet tip. The samples were placed in 2 mL eppendorf tubes from which 10-fold dilutions were prepared using RK-13 growth media. Samples were diluted from initial spore concentrations of 105 to 100/ mL in 900 mL of growth media. Each of the dilutions was then pipetted in 150 ml aliquots onto 6 replicate wells of 2 day old RK-13 monolayers grown in Lab Tech-II chamber slides (Nalgene Nunc, Naperville, Ill) that showed 85–90% confluent cell growth. The two uninfected wells on each slide served as negative controls. Sample processing and cell culture inoculation occurred within 1 h of UV exposure. 2.6. Cell culture analysis UV treated samples and controls were analyzed using a modified foci detection method-most probable number (FDM-MPN) cell culture technique [7,8]. Modifications included extending the incubation period from 2 days post infection (as performed for Cryptosporidium) to 5 days for microsporidia. On the fifth day post infection, the well chamber slides were removed from the incubator, the media aspirated and the upper chamber portion removed. The slides were then fixed for 10 min in 100% methanol (Sigma, St. Louis, MO) and allowed to air dry. The slides were then stained in 0.05% solution of Calcofluor White M2R in 0.1 M phosphate buffered saline (pH 7.0–8.0) (Sigma, St. Louis, MO) for 15 s and counter stained in 0.2% Evans Blue for 5 s [7]. After the slides were dry, cover slips were attached using nail polish and the slides were placed in the dark at room temperature until microscopic evaluation was performed. Stained monolayers were evaluated using epifluorescence microscopy (excitation filter 340–380 nm) at 200 and confirmed at 400 magnification. Each well on the chamber slide was viewed and scored as either positive (infection present) or negative (no infection). When infection was present, large clusters of infectious E. intestinalis spores were easily identified against the dark blue background of uninfected RK-13 cells. The MPN of infectious spores was determined using the Information Collection Rule (ICR) Most Probable Number calculator, version 1.00 [9].
3. Results and discussion Replicate cell culture analyses were performed using three UV doses for both low- and medium-pressure UV (Fig. 1). The control spores of E. intestinalis had a mean percent infectivity of 2.1%70.3% with an average MPN/mL of 8.1 103 and a standard deviation of 2.1 103 based upon an average initial concentration of 3.8 105/mL74.0 104 spores/mL. The spores that were exposed to either low- or medium-pressure UV
Fig. 1. Low- and medium-pressure UV inactivation of Microsporidia E. intestinalis. Error bars indicate 95% confidence intervals.
doses of 6 and 9 mJ/cm2 showed no infectious foci in any of the cell monolayers yielding >3.9–>4.0 log10 inactivation of E. intestinalis. A low- or medium-pressure dose of 3 mJ/cm2 showed an inactivation of 1.6–2.0 log10. Previous studies using the FDM-MPN to evaluate the effectiveness of low- and medium-pressure UV for the inactivation of Cryptosporidium parvum showed slightly higher oocyst inactivation at a dose of 3 mJ/cm2 (2.0– 2.9 log10) [10]. Previous studies of low- and mediumpressure UV light inactivation of C. parvum using mouse infectivity analysis have shown tailing in the UV inactivation curve at doses >25 mJ/cm2 [6]. This tailing was not noted in previous FDM-MPN assays for Cryptosporidium post UV exposure [10]. Replicate experiments showed minimal change in the infectivity of the stock culture of E. intestinalis throughout the time frame of the study. All of the microsporidia stocks were o30 days of age at the time of assay and the TCID50, infectious dose at which 50% of the tissue culture wells were positive, was 55 spores. Previous dose response data for E. intestinali in a cell culture system reported a TCID50 of 915 spores [11]. The potential use of UV disinfection for the inactivation of protozoa in drinking water has been steadily growing since it was determined that both medium and low-pressure UV could inactivate Cryptosporidium oocysts and Giardia cysts at doses that are easily achievable for drinking water treatment. Microsporidia has been placed on the EPAs candidate contaminate list as potentially requiring regulation and monitoring. This research demonstrates that UV light at dosages utilized for drinking water treatment is capable of achieving high levels of inactivation of microsporidia E. intestinalis.
Acknowledgements This work was supported by Calgon Carbon Corporation, Pittsburgh, PA. At the time of the research,
3164
D.E. Huffman et al. / Water Research 36 (2002) 3161–3164
Dr. B. Dussert was the Director of UV Research at Calgon Carbon Corp. [7]
References [1] Bryan RT. Microsporidiosis as an AIDS-related opportunistic infection. Clin Infect Dis 1995;21(1):62–5. [2] Cotte L, Rabodonirina M, Chapuis F, Bailly F, Bissuel F, Raynal C, Gelas P, Persat F, Piens M-A, Trepo C. Waterborne outbreak of intestinal microsporidiosis in persons with and without human immunodeficiency virus infection. J Infect Dis 1999;180:2003–8. [3] Shadduck JA, Polley MB. Some factors influencing the invitro infectivity and replication of Encephalitozoon cuniculi. J Protozool 1978;25:491–6. [4] Shadduck JA. Human microsporidiosis and AIDS. Rev Infect Dis 1989;11:203–7. [5] Waller T. Sensitivity of Encephalitozoon cuniculi to various temperatures disinfectants and drugs. Lab Anim 1979;13:227–30. [6] Craik SA, Weldon D, Finch GR, Bolton JR, Belosevic M. Inactivation of Cryptosporidium parvum oocysts using
[8]
[9]
[10]
[11]
medium- and low-pressure ultraviolet radiation. Water Res 2001;35(6):1387–98. John D. Masters thesis: Effects of Chemical Disinfection on Microsporidia E. Intestinalis. University of Arizona Department of Soil and Water Science, Tucson, Arizona, 2000. Slifko TR, Huffman DE, Rose JB. A most-probable number assay for enumeration of infectious Cryptosporidium parvum oocysts. Appl Environ Microbiol 1999;65(9):3936–41. Klee AJ. ICR most probable number calculator, Version 1.00 [online], Risk Reduction Laboratory, US Environmental Protection Agency, Cincinnati, OH, 1999. http:// www.epa.gov/nerlcwww/other.htm Huffman DE. Unpublished data. University of South Florida, College of Marine Science 140 7th Ave. South, St. Petersburg, FL 33701, 2001. Wolk DM, Johnson CH, Rice EW, Marshall MM, Grahn KF, Plummer CB, Sterling CR. A spore counting method and cell culture model for chlorine disinfection studies of Encephalitozoon syn. Septata intestinalis. Appl Environ Microbiol 2000;66(4):1266–73.