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Parasitology Research

, Volume 101, Issue 3, pp 569–575 | Cite as

Human-virulent microsporidian spores in solid waste landfill leachate and sewage sludge, and effects of sanitization treatments on their inactivation

  • Thaddeus K. GraczykEmail author
  • Malgorzata Kacprzak
  • Ewa Neczaj
  • Leena Tamang
  • Halshka Graczyk
  • Frances E. Lucy
  • Autumn S. Girouard
Original Paper

Abstract

Solid waste landfill leachate and sewage sludge samples were quantitatively tested for viable Enterocytozoon bieneusi, Encephalitozoon intestinalis, Encephalitozoon hellem, and Encephalitozoon cuniculi spores by the multiplexed fluorescence in situ hybridization (FISH) assay. The landfill leachate samples tested positive for E. bieneusi and the sludge samples for E. bieneusi and E. intestinalis. The effects of four sanitization treatments on the inactivation of these pathogens were assessed. Depending on the variations utilized in the ultrasound disintegration, sonication reduced the load of human-virulent microsporidian spores to nondetectable levels in 19 out of 27 samples (70.4%). Quicklime stabilization was 100% effective, whereas microwave energy disintegration was 100% ineffective against the spores of E. bieneusi and E. intestinalis. Top-soil stabilization treatment gradually reduced the load of both pathogens, consistent with the serial dilution of sewage sludge with the soil substrate. This study demonstrated that sewage sludge and landfill leachate contained high numbers of viable, human-virulent microsporidian spores, and that sonication and quicklime stabilization were the most effective treatments for the sanitization of sewage sludge and solid waste landfill leachates. Multiplexed FISH assay is a reliable quantitative molecular fluorescence microscopy method for the simultaneous identification of E. bieneusi, E. intestinalis, E. hellem, and E. cuniculi spores in environmental samples.

Keywords

Sewage Sludge Sludge Sample Landfill Leachate Quicklime Sewage Sludge Sample 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgments

The study was supported by the Organization for Economic Co-Operation and Development (grant no. AGR/PR20061), the Fulbright Senior Specialist Fellowship (grant no. 2225 to Graczyk), Johns Hopkins Center in Urban Environmental Health (grant no. P30 ES03819), Alternatives Research & Development Foundation, NOAA Chesapeake Bay Office (grant no. NA04NMF4570426), Procter & Gamble Foundation, and Johns Hopkins Center for a Livable Future.

References

  1. Amar CFL, East CL, Grant KA, Gray J, Itrriza-Gomara M, Maclure EA, McLauchlin J (2005) Detection of viral, bacterial, and parasitological RNA or DNA of nine intestinal pathogens in fecal samples archived as part of the English infectious intestinal disease study—assessment of the stability of target nucleic acid. Diagn Mol Pathol 14:90–96PubMedCrossRefGoogle Scholar
  2. Ash LR, Orihel TC (1987) Parasites: a guide to laboratory procedures and identification. American Society of Clinical Pathologists (ASCP) Press, Chicago, IllinoisGoogle Scholar
  3. Ashokkumar M, Vu T, Grieser F, Weerawardena A, Anderson N, Pilkinbton N, Dixon DR (2003) Ultrasonic treatment of Cryptosporidium oocysts. Water Sci Technol 47:173–177PubMedGoogle Scholar
  4. Bednarska M, Bajer A, Sinski E, Girouard AS, Tamang L, Graczyk TK (2007) Fluorescent in situ hybridization as a tool to retrospectively identify Cryptosporidium parvum and Giardia lamblia in samples from terrestrial mammalian wildlife. Parasitol Res 100:455–460PubMedCrossRefGoogle Scholar
  5. Bern C, Kawai V, Vargas D, Rabke-Vearni J, Williamson J, Chavez-Valdez R, Xiao L, Sulaiman I, Vivar A, Ticona E, Navincopa M, Cama V, Moura H, Secor WW, Visvesvara G, Gilman RH (2005) The epidemiology of intestinal microsporidiosis in patients with HIV/AIDS in Lima, Peru. J Infect Dis 191:1658–1664PubMedCrossRefGoogle Scholar
  6. Bouchet F, Boulard Y (1991) Ultrastructural changes following treatment with a microwave pulse in the oocyst of Eimeria magna Penard, 1925. Parasitol Res 77:585–589PubMedCrossRefGoogle Scholar
  7. Chauret C, Springthorpe S, Sattar S (1999) Fate of Cryptosporidium oocysts, Giardia cysts, and microbial indicators during wastewater treatment and anaerobic sludge digestion. Can J Microbiol 45:257–262PubMedCrossRefGoogle Scholar
  8. Collins MV, Flick GJ, Smith SA, Fayer R, Rubendall R, Lindsay DS (2005) The effect of E-beam irradiation and microwave energy on Eastern oysters (Crasssostrea virginica) experimentally infected with Cryptosporidium parvum. J Eukaryot Microbiol 52:484–488PubMedCrossRefGoogle Scholar
  9. Conteas CN, Berlin OGW, Ash LR, Pruthi JS (2000) Therapy for human gastrointestinal microsporidiosis. Am J Trop Med Hyg 63:121–127PubMedGoogle Scholar
  10. Cotte L, Rabodonirina M, Chapuis F, Bailly F, Bissuel F, Raynal C, Gelas P, Persat F, Piens MA, Treppo C (1999) Waterborne outbreak of intestinal microsporidiosis in persons with and without human immunodeficiency virus infection. J Infect Dis 180:2003–2008PubMedCrossRefGoogle Scholar
  11. da Silva AJ, Schwartz DA, Visvesvara GS, de Moura H, Slemenda SB, Pieniazek NJ (1996) Sensitive PCR diagnosis of infections by Enterocytozoon bieneusi (Microsporidia) using primers based on the region coding for small-subunit rRNA. J Clin Microbiol 34:986–987PubMedGoogle Scholar
  12. Didier ES (2005) Microsporidiosis: an emerging and opportunistic infection in humans and animals. Acta Trop 94:61–76PubMedCrossRefGoogle Scholar
  13. Didier ES, Stovall ME, Green LC, Brindley PJ, Sestak K, Didier PJ (2004) Epidemiology of microsporidiosis: source and modes of transmission. Vet Parasitol 126:145–166PubMedCrossRefGoogle Scholar
  14. Didier ES, Maddry JA, Brindley PJ, Stovall ME, Didier PJ (2005) Therapeutic strategies for human microsporidia infections. Expert Rev Anti Infect Ther 3:419–434PubMedCrossRefGoogle Scholar
  15. Dowd SE, Gerba CP, Pepper IL (1998) Confirmation of the human-pathogenic microsporidia Enterocytozoon bieneusi, Encephalitozoon intestinalis, and Vittaforma corneae in water. Appl Environ Microbiol 64:3332–3335PubMedGoogle Scholar
  16. Enriquez FJ, Taren D, Cruz-Lopez A, Muramoto M, Palting JD, Cruz P (1998) Prevalence of intestinal encephalitozoonosis in Mexico. Clin Infect Dis 26:1227–1229PubMedCrossRefGoogle Scholar
  17. Environmental Protection Agency (EPA) (1998) Announcement of the drinking water contaminant candidate list: notice. Fed Regist 63:10272–10287Google Scholar
  18. Fournier S, Liguory O, Santillana-Hayat M, Guillot E, Sarfari C, Dumoutier N, Molina J, Derouin F (2000) Detection of microsporidia in surface water: a one-year follow-up study. FEMS Immunol Med Microbiol 29:95–100PubMedCrossRefGoogle Scholar
  19. Gale P (2005) Land application of treated sewage sludge: quantifying pathogen risk from consumption of crops. J Appl Microbiol 98:380–396PubMedCrossRefGoogle Scholar
  20. Gerba CP, Riley KR, Nwachcuku N, Ryu H, Abbaszadegan M (2003) Removal of Encephalitozoon intestinalis, calicivirus, and coliphages by conventional drinking water treatment. J Environ Sci Health A Tox Hazard Subst Environ Eng 38:1259–1268PubMedGoogle Scholar
  21. Graczyk TK, Cranfield MR (2000) Cryptosporidium serpentis oocysts and microsporidian spores in stools of captive snakes. J Parasitol 86:413–414PubMedGoogle Scholar
  22. Graczyk TK, Bosco-Nizeyi J, da Silva AJ, Moura INS, Pieniazek NJ, Cranfield MR, Lindquist HD (2002) A single genotype of Encephalitozoon intestinalis infects free-ranging gorillas and people sharing their habitats, Uganda. Parasitol Res 88:926–931PubMedCrossRefGoogle Scholar
  23. Graczyk TK, Conn DB, Lucy F, Minchin D, Tamang L, Moura LNS, da Silva AJ (2004) Human waterborne parasites in zebra mussels (Dreissena polymorpha) from the Shannon River drainage, Ireland. Parasitol Res 93:389–391CrossRefGoogle Scholar
  24. Graczyk TK, Girouard AS, Tamang L, Nappier SP, Schwab KJ (2006) Recovery, bioaccumulation, and inactivation of human waterborne pathogens by the Chesapeake Bay non-native oyster, Crassostrea ariakensis. Appl Environ Microbiol 72:3390–3395PubMedCrossRefGoogle Scholar
  25. Graczyk TK, Lewis EJ, Glass G, da Silva AJ, Tamang L, Girouard AS, Curriero FC (2007) Quantitative assessment of viable Cryptosporidium parvum load in commercial oysters (Crassostrea virginica) in the Chesapeake Bay. Parasitol Res 100:247–253PubMedCrossRefGoogle Scholar
  26. Hester FD, Lindquist HD, Bobst AM, Schaffer FW (2000) Fluorescent in situ detection of Encephalitozoon hellem spores with a 6-carboxyfluorescein-labeled ribosomal RNA-targeted oligonucleotide probe. J Eukaryot Microbiol 47:299–308PubMedCrossRefGoogle Scholar
  27. Hutchison ML, Walters LD, Moore A, Avery SM (2005) Declines of zoonotic agents in liquid livestock wastes stored in batches on-farm. J Appl Microbiol 99:58–65PubMedCrossRefGoogle Scholar
  28. John DE, Nwachcuku N, Pepper IL, Gerba CP (2003) Development and optimization of a quantitative cell culture infectivity assay for the microsporidium Encephalitozoon intestinalis and application to ultraviolet light inactivation. J Microbiol Methods 52:183–196PubMedCrossRefGoogle Scholar
  29. Matchis A, Weber R, Deplazes P (2005) Zoonotic potential of microsporidia. Clin Microbiol Rev 18:423–445CrossRefGoogle Scholar
  30. Muller A, Bialek R, Kamper R, Fatkenheuer G, Salzberger B, Franzen C (2001) Detection of microsporidia in travelers with diarrhea. J Clin Microbiol 39:1630–1632PubMedCrossRefGoogle Scholar
  31. Nwachcuku N, Gerba CP (2004) Emerging waterborne pathogens: can we kill them all? Curr Opin Biotechnol 15:175–180PubMedCrossRefGoogle Scholar
  32. Slifko TR, Smith HV, Rose JB (2000) Emerging parasite zoonoses associated with water and food. Int J Parasitol 30:1379–1393PubMedCrossRefGoogle Scholar
  33. Slodkowicz-Kowalska A, Graczyk TK, Tamang L, Jedrzejewski S, Nowosad A, Zduniak P, Solarczyk P, Girouard AS, Majewska AC (2006) Microsporidia species known to infect humans are present in aquatic birds: implications for transmission via water? Appl Environ Microbiol 72:4540–4544PubMedCrossRefGoogle Scholar
  34. Sobsey M (1978) Field survey of enteric viruses in solid waste landfill leachates. Am J Publ Health 68:858–864CrossRefGoogle Scholar
  35. Sparfel JM, Sarfati C, Liquory O, Caroff B, Dumoutier N, Gueglio B, Billaud E, Raffi F, Molina LM, Miegeville M, Derouin F (1997) Detection of microsporidia and identification of Enterocytozoon bieneusi in surface water by filtration followed by specific PCR. J Eukaryot Microbiol 44:78SPubMedCrossRefGoogle Scholar
  36. Straub TM, Pepper IL, Gerba CP (1993) Hazard from pathogenic microorganisms and land-disposed sewage sludge. Rev Environ Contam Toxicol 132:55–91PubMedGoogle Scholar
  37. Szostakowska B, Kruminis-Lozowska W, Racewicz M, Knigh R, Tamang L, Myjak P, Graczyk TK (2004) Cryptosporidium parvum and Giardia lamblia recovered from flies on a cattle farm and in a landfill. Appl Environ Microbiol 70:3742–3744PubMedCrossRefGoogle Scholar
  38. Thurston-Enriquez J, Watt AP, Dowd SE, Enriquez R, Pepper IL, Gerba CP (2002) Detection of protozoan parasites and microsporidia in irrigation waters used for crop production. J Food Prot 65:378–382PubMedGoogle Scholar
  39. Velasques JN, Carnevale S, Labbe JH, Chertcoff A, Cabrera G, Oelemann W (1999) In situ hybridization: a molecular approach for the diagnosis of the microsporidian parasite Enterocytozoon bieneusi. Hum Pathol 30:54–58CrossRefGoogle Scholar
  40. Vincek V, Mehdi N, Block N, Welsh CF, Mehrdad N, Morales AR (2005) Methodology for preservation of high molecular-weight RNA in paraffin-embedded tissue-application for laser-capture microdissection. Diagn Mol Pathol 14:127–133PubMedCrossRefGoogle Scholar
  41. Weber R, Bryan RT (1994) Microsporidial infections in immunodeficient and immunocompetent patients. Clin Infect Dis 19:517–521PubMedGoogle Scholar
  42. Weber R, Bryan RT, Schwartz DA, Owen RL (1994) Human microsporidial infections. Clin Microbiol Rev 7:426–461PubMedGoogle Scholar
  43. Weiss LM (2001) Microsporidia: emerging pathogenic protists. Acta Trop 78:89–102PubMedCrossRefGoogle Scholar
  44. Westrell T, Schonning C, Stenstrom TA, Ashbolt NJ (2004) QMRA (quantitative microbial risk assessment) and HACCP (hazard analysis and critical control points) for management of pathogens in wastewater and sewage sludge treatment and reuse. Water Sci Technol 50:23–30PubMedGoogle Scholar
  45. Wolk DM, Johnson CH, Rice EW, Marshall MM, Grahn KF, Plumer CB, Sterling R (2000) A spore counting method and cell culture model for chlorine disinfection studies of Encephalitozoon intestinalis syn Septata intestinalis. Appl Environ Microbiol 66:1266–1273PubMedCrossRefGoogle Scholar
  46. Yangin C, Yilmaz S, Altinbas M, Ozturk I (2002) A new process for the combined treatment of municipal wastewaters and land leachates in coastal areas. Water Sci Technol 46:111–118PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Thaddeus K. Graczyk
    • 1
    • 2
    • 3
    Email author
  • Malgorzata Kacprzak
    • 4
  • Ewa Neczaj
    • 4
  • Leena Tamang
    • 1
  • Halshka Graczyk
    • 5
  • Frances E. Lucy
    • 6
  • Autumn S. Girouard
    • 3
  1. 1.Department of Environmental Health Sciences, Division of Environmental Health EngineeringJohns Hopkins Bloomberg School of Public HealthBaltimoreUSA
  2. 2.Johns Hopkins Water and Public Health CenterJohns Hopkins Bloomberg School of Public HealthBaltimoreUSA
  3. 3.Department of Molecular Microbiology and Immunology, Bloomberg School of Public HealthJohns Hopkins UniversityBaltimoreUSA
  4. 4.Institute of Environmental Health EngineeringCzestochowa University of TechnologyCzestochowaPoland
  5. 5.Johns Hopkins UniversityBaltimoreUSA
  6. 6.School of ScienceInstitute of TechnologySligoIreland

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