Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Johnson, C. H. Articles by Korich, D. G. Search for Related Content PubMed PubMed Citation Articles by Johnson, C. H. Articles by Korich, D. G. Agricola Articles by Johnson, C. H. Articles by Korich, D. G.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, February 2003, p. 1325-1326, Vol. 69, No. 2
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.2.1325-1326.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Chlorine Inactivation of Spores of Encephalitozoon spp.

C. H. Johnson,^1* M. M. Marshall,² L. A. DeMaria,¹ J. M. Moffet,² and D. G. Korich²

U.S. Environmental Protection Agency, Cincinnati, Ohio 45268,¹ Sterling Parasitology Laboratory, University of Arizona, Tucson, Arizona 85721²

Received 27 February 2002/ Accepted 13 November 2002

Top Abstract Introduction References

 
This report is an extension of a preliminary investigation on the use of chlorine to inactivate spores of Encephalitozoon intestinalis and to investigate the effect of chlorine on two other species, E cuniculi and E. hellem, associated with human infection. The 50% tissue culture infective doses of these three species were also determined. On the basis of the results obtained, it appears that chlorination of water is an effective means of controlling spores of these organisms in the aquatic environment.

Top Abstract Introduction References

 
The phylum Microspora is composed of a diverse group of protistan parasites that infect a wide range of vertebrate and invertebrate hosts. Certain species of these obligate intracellular parasites are etiological agents of microsporidiosis in humans. While immunocompetent individuals may become infected, microsporidiosis is a particularly devastating disease in immunosuppressed individuals, especially those infected with the human immunodeficiency virus. The infective, spore stage of these organisms is released into the environment from infected hosts. By using molecular procedures, several studies have detected these organisms in water (3, 6). Recently, a report on a retrospective study of an outbreak of intestinal microsporidiosis implicated water as the probable vehicle of transmission (2). Interest in the possible role of these parasites as waterborne pathogens has prompted research into the efficacy of chlorination for controlling these organisms in water. This report is an extension of a preliminary investigation on the use of chlorine to inactivate spores of Encephalitozoon intestinalis (7) and to investigate the effect of chlorine on two other species, E. cuniculi and E. hellem, associated with human infection. The 50% tissue culture infective doses (TCID₅₀) of these three species were also determined.

E. cuniculi (ATCC 50502), E. hellem (ATCC 50451), and E. intestinalis (ATCC 50603) were propagated in monolayers of RK-13 (ATCC CCL-37) rabbit kidney cells incubated at 35°C in a humid atmosphere of 5% CO₂ as previously described (7). The spores were harvested by removing the culture medium from the cell monolayers and concentrated by centrifugation at 1,000 x g for 10 min. The supernatant was removed by aspiration, and the spore pellet was further purified by differential density gradient centrifugation by using an isopycnic Percoll (Amersham Pharmacia Biotech, Piscataway, N.J.) gradient and a final wash step using reagent grade water (7). The concentration of spores was determined by counting with a hemacytometer, and the stock preparations were stored at 4°C in reagent grade water. Spore preparations were used within 1 week of harvesting. Spores from individual species were used in the inactivation experiments.

TCID₅₀s were determined by using a logit dose-response model (5) to describe the dose-response relationship between the tissue culture wells and the Encephalitozoon sp. spores. Briefly, the response logit (RL) value was calculated for each inoculum of spores as the natural logarithm (ln) of the proportion of infected tissue culture wells (P) divided by 1 minus the proportion of inoculated wells {RL = ln[P/(1 - P)]}. The RL values were treated as dependent variables (y) for linear regression analysis with the logarithm (log₁₀) of the number of spores in each dose serving as the independent variable (x). Regression analysis was then used to perform a least-squares regression to calculate the regression model and parameters. The logit models were used to calculate the TCID₅₀ for each species and to determine the number of infective spores remaining in each inoculum after disinfectant treatment.

Inactivation experiments were conducted at 23 ± 2°C in chlorine demand-free (CDF) 0.05 M potassium dihydrogen phosphate buffer adjusted to pH 7.0 as previously reported (4). The reaction vessels used were 400-ml beakers containing 90 ml of CDF buffer. Reagent grade sodium hypochlorite was added to each beaker to achieve the desired level of chlorine prior to the addition of spores. An inoculum (10 ml) containing 10⁷ spores was added to each reaction vessel at time zero. During the course of the experiments, the reaction vessel contents were continuously mixed with a magnetic stirring device. Ten-milliliter samples were removed from the reaction vessels at the desired exposure times, and the chlorine was immediately neutralized by the addition of 0.1 ml of 10% (wt/vol) sodium thiosulfate. Chlorine levels were determined initially and at each exposure time by the N,N-dimethyl-p-phenylenediamine colorimetric method (1). Vessels containing CDF buffer without chlorine served as controls for the unexposed spores and were treated in the same manner as the chlorine-exposed samples. Addition of sodium thiosulfate to the control vessel did not have a deleterious effect on the spores. The logit dose-response regression model (5) was used to determine the number of viable spores after chlorine treatment. The log₁₀ reduction (LR) was determined by subtracting the number (log₁₀) of viable spores remaining after treatment (N) from the total number (log₁₀) of spores in the initial inoculum (N₀) (LR = log₁₀N - log₁₀N₀).

The TCID₅₀ shown in Table 1 were determined by using the mean number of infected tissue culture wells at the various dilutions. Since the logit expressions for 0 and 100% infectivity are undefined, they were not used in the calculations. The calculated TCID₅₀ for E. cuniculi was 15 spores; for E. hellem, it was 68 spores, and for E. intestinalis, it was 27 spores. The results of the chlorine inactivation experiments are shown in Table 2. The chlorine level at the beginning of each experiment was 2.5 ± 0.2 mg liter^-1. The spore preparations exerted an initial rapid chlorine demand, and the residual chlorine level at the end of each exposure time was 2.0 ± 0.2 mg liter^-1. E. hellem showed a greater degree of resistance than the other two species at the shorter exposure times. However, all three species were inactivated by 4 orders of magnitude or more after an exposure time of 6 to 8 min, equating to a simple Ct value (Ct = disinfectant concentration [milligrams per liter] x exposure time [minutes]) of 16 mg-min liter^-1.

View this table: [in this window] [in a new window]

TABLE 1. Infectivity of spores of Encephalitozoon spp. inoculated on monolayers of rabbit kidney cells (RK-13)

View this table: [in this window] [in a new window]

TABLE 2. Chlorine inactivation of spores of Encephalitozoon spp.a

The TCID₅₀ for E. cuniculi and E. hellem reported here are the first published values for these species. The currently reported TCID₅₀ of 27 spores for E. intestinalis is considerably lower than the previously reported value of 915 (7). The same procedure and reagents were used in both studies, with the only variable being the serum, which was obtained from a different vendor for the present study. This finding underscores the variability that can occur in the determination of TCID₅₀ and the necessity of including individual control cultures for each organism when conducting inactivation experiments. These results confirm the previous report of Wolk et al. (7) showing that spores of E. intestinalis are sensitive to chlorination and further indicate that other species of Encephalitozoon are also capable of being inactivated by chlorine. The level of chlorination (Ct = 16) required to inactivate spores of Encephalitozoon spp. is readily attainable by most water utilities in the United States (4). On the basis of these results, it appears that chlorination of water is an effective means of controlling spores of these organisms in the aquatic environment.

 
* Corresponding author. Mailing address: U.S. Environmental Protection Agency, 26 W. M. L. King Dr., MS-387, Cincinnati, OH 45268. Phone: (513) 569-7345. Fax: (513) 569-7328. E-mail: johnson.cliff{at}epa.gov.

Top Abstract Introduction References

 

1
1. American Public Health Association. 1998. Standard methods for the examination of water and wastewater, 20th ed. American Public Health Association, Washington, D.C.
2
2. Cotte, L., M. Rabodonirina, F. Chapuis, F. Bailly, F. Bissuel, C. Raynal, P. Gelas, F. Persat, M. Piens, and C. Trepo. 1999. Waterborne outbreak of intestinal microsporidiosis in persons with and without human immunodeficiency virus infection. J. Infect. Dis. 180:2003-2008.
3
3. Dowd, S. E., C. P. Gerba, and I. L. Pepper. 1998. Confirmation of the human-pathogenic microsporidia Enterocytozoon bieneusi, Encephalitozoon intestinalis, and Vittaforma cornea in water. Appl. Environ. Microbiol. 64:3332-3335.[Abstract/Free Full Text]
4
4. Johnson, C. H., E. W. Rice, and D. J. Reasoner. 1997. Inactivation of Helicobacter pylori by chlorination. Appl. Environ. Microbiol. 63:4969-4970.[Abstract]
5
5. Korich, D. G., M. M. Marshall, H. V. Smith, J. O'Grady, Z. Bukhari, C. R. Fricker, J. P. Rosen, and J. L. Clancy. 2000. Inter-laboratory comparison of the CD-1 neonatal logistic dose-response model for Cryptosporidium parvum oocysts. J. Eukaryot. Microbiol. 47:294-298.[CrossRef][Medline]
6
6. Sparfel, J. M., C. Sarfati, O. Liguory, B. Caroff, N. Dumoutier, B. Gueglio, E. Billaud, F. Raffi, J. Molina, M. Miegeville, and F. Derouin. 1997. Detection of microsporidia and identification of Enterocytozoon bieneusi in surface water by filtration followed by specific PCR. J. Microbiol. 44:78S.
7
7. Wolk, D. M., C. H. Johnson, E. W. Rice, M. M. Marshall, K. F. Grahn, C. B. Plummer, and C. R. Sterling. 2000. A spore counting method and cell culture model for chlorine disinfection studies of Encephalitozoon syn. Septata intestinalis. Appl. Environ. Microbiol. 66:1266-1273.[Abstract/Free Full Text]

---

Applied and Environmental Microbiology, February 2003, p. 1325-1326, Vol. 69, No. 2
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.2.1325-1326.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Mathis, A., Weber, R., Deplazes, P. (2005). Zoonotic Potential of the Microsporidia. Clin. Microbiol. Rev. 18: 423-445 [Abstract] [Full Text]  
• Hoffman, R. M., Marshall, M. M., Polchert, D. M., Helen Jost, B. (2003). Identification and Characterization of Two Subpopulations of Encephalitozoon intestinalis. Appl. Environ. Microbiol. 69: 4966-4970 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Johnson, C. H. Articles by Korich, D. G. Search for Related Content PubMed PubMed Citation Articles by Johnson, C. H. Articles by Korich, D. G. Agricola Articles by Johnson, C. H. Articles by Korich, D. G.