Abstract
A detection method was developed and assessed for the sensitive recovery of microsporidia from livestock fecal and manure-impacted environmental samples. Sensitive recovery of microsporidia was achieved when samples were subjected to 1) purification by sucrose floatation, 2) DNA extraction using the Qiagen QIAamp DNA Stool Mini Kit, 3) polymerase chain reaction (PCR) analysis using generic primers for microsporidia, and 4) DNA sequence analysis to identify which microsporidia were present in each sample. Livestock fecal and wastewater samples were inoculated with 1,000 and 100 Encephalitozoon intestinalis spores/g or ml of feces or wastewater. For cattle wastewater, ten of ten replicates were positive by PCR at concentrations of 1,000 spores/ml, and two of ten replicates were positive at concentrations of 100 spores/ml. For swine wastewater, ten of ten replicates were positive at concentrations of 1,000 spores/ml, and four of ten replicates were positive at concentrations of 100 spores/ml. For cattle feces, three of ten replicates were positive at the concentration of 1,000 spores/g. Several environmental samples were screened using this method, with two of 34 samples positive for human pathogenic microsporidia. To our knowledge, this is the first report of Encephalitozoon cuniculi detection in swine feces and wastewater.
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References
Akerstedt J (2002) An indirect ELISA for detection of Encephalitozoon cuniculi infection in farmed blue foxes (Alopex lagopus). Acta Vet Scand 43:211–220
Avery SW, Undeen AH (1987) The isolation of microsporidia and other pathogens from concentrated ditch water. J Am Mosq Control Assoc 3:54–58
Bornay-Llinares FJ, da Silva AJ, Moura H, Schwartz DA, Visvesvara GS, Pieniazek NJ, Cruz-Lopez A, Hernandez-Jauregui P, Guerrero J, Enriquez FJ (1998) Immunologic, microscopic, and molecular evidence of Encephalitozoon intestinalis (Septata intestinalis) infection in mammals other than humans. J Infect Dis 178:820–826
Breitenmoser AC, Mathis A, Burgi E, Weber R, Deplazes P (1999) High prevalence of Enterocytozoon bieneusi in swine with four genotypes that differ from those identified in humans. Parasitology 118:447–453
Buckholt MA, Lee JH, Tzipori S (2002) Prevalence of Enterocytozoon bieneusi in swine: an 18-month survey at a slaughterhouse in Massachusetts. Appl Environ Microbiol 68:2595–2599
Cotte L, Rabodonirina M, Chapuis F, Bailly F, Bissuel F, Raynal C, Gelas P, Persat F, Piens MA, Trepo C (1999) Waterborne outbreak of intestinal microsporidiosis in persons with and without human immunodeficiency virus infection. J Infect Dis 180:2003–2008
Del Aguila C, Izquierdo F, Navajas R, Pieniazek NJ, Miro G, Alonso AI, da Silva AJ, Fenoy S (1999) Enterocytozoon bieneusi in animals: rabbits and dogs as new hosts. J Eukaryot Microbiol 46:8S–9S
Dengjel BM, Zahler M, Hermanns W, Heinritzi K, Spillmann T, Thomschke A, Loscher T, Gothe R, Rinder H (2001) Zoonotic potential of Enterocytozoon bieneusi. J Clin Microbiol 39:4495–4499
Deplazes P, Mathis A, Muller C, Weber R (1996) Molecular epidemiology of Encephalitozoon cuniculi and first detection of Enterocytozoon bieneusi in faecal samples of pigs. J Eukaryot Microbiol 43:S93
Didier ES, Stovall ME, Green LC, Brindley PJ, Sestak K, Didier PJ (2004) Epidemiology of microsporidiosis: sources and modes of transmission. Vet Parasitol 126:145–166
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 64:3332–3335
Dowd SE, Gerba CP, Kamper M, Pepper IL (1999) Evaluation of methodologies including immunofluorescent assay (IFA) and the polymerase chain reaction (PCR) for detection of human pathogenic microsporidia in water. J Microbiol Methods 35:43–52
Dowd SE, John D, Eliopolus J, Gerba CP, Naranjo J, Klein R, Lopez B, de Mejia M, Mendoza CE, Pepper IL (2003) Confirmed detection of Cyclospora cayetanesis, Encephalitozoon intestinalis and Cryptosporidium parvum in water used for drinking. J Water Health 1:117–123
EPA (1998) Announcement of the drinking water contaminant candidate list. Fed Regist 63:10273–10287
Fayer R, Santin M, Palmer R (2003a) Comparison of microscopy and PCR for detection of three species of Encephalitozoon in feces. J Eukaryot Microbiol 50:572–573
Fayer R, Santin M, Palmer R, Li XD (2003b) Detection of Encephalitozoon hellem in feces of experimentally infected chickens. J Eukaryot Microbiol 50:574–575
Fayer R, Santin M, Trout JM (2003c) First detection of microsporidia in dairy calves in North America. Parasitol Res 90:383–386
Fedorko DP, Nelson NA, Cartwright CP (1995) Identification of microsporidia in stool specimens by using PCR and restriction endonucleases. J Clin Microbiol 33:1739–1741
Fournier S, Liguory O, Santillana-Hayat M, Guillot E, Sarfati C, Dumoutier N, Molina JM, Derouin F (2000) Detection of microsporidia in surface water: a one-year follow-up study. FEMS Immunol Med Microbiol 29:95–100
Franzen C, Muller A (1999) Cryptosporidia and microsporidia—waterborne diseases in the immunocompromised host. Diagn Microbiol Infect Dis 34:245–262
Halanova M, Letkova V, Macak V, Stefkovic M, Halan M (1999) The first finding of antibodies to Encephalitozoon cuniculi in cows in Slovakia. Vet Parasitol 82:167–171
Hollister WS, Canning EU, Viney M (1989) Prevalence of antibodies to Encephalitozoon cuniculi in stray dogs as determined by an ELISA. Vet Rec 124:332–336
KatzwinkelWladarsch S, Lieb M, Heise W, Loscher T, Rinder H (1996) Direct amplification and species determination of microsporidian DNA from stool specimens. Trop Med Int Health 1:373–378
Kuczynska E, Shelton DR (1999) Method for detection and enumeration of Cryptosporidium parvum oocysts in feces, manures, and soils. Appl Environ Microbiol 65:2820–2826
Li X, Palmer R, Trout JM, Fayer R (2003a) Infectivity of microsporidia spores stored in water at environmental temperatures. J Parasitol 89:185–188
Li X, Tate KW, Dunbar LA, Huang B, Atwill ER (2003b) Efficiency for recovering Encephalitozoon intestinalis spores from waters by centrifugation and immunofluorescence microscopy. J Eukaryot Microbiol 50:579–580
Lobo ML, Teles A, Da Cunha MB, Henriques J, Lourenco AM, Antunes F, Matos O (2003) Microsporidia detection in stools from pets and animals from the zoo in Portugal: a preliminary study. J Eukaryot Microbiol 50:581–582
Lores B, del Aguila C, Arias C (2002) Enterocytozoon bieneusi (Microsporidia) in faecal samples from domestic animals from Galicia, Spain. Mem Inst Oswaldo Cruz 97:941–945
Mathis A, Breitenmoser AC, Deplazes P (1999) Detection of new Enterocytozoon genotypes in faecal samples of farm dogs and a cat. Parasite 6:189–193
Muller-Doblies UU, Herzog K, Tanner I, Mathis A, Deplazes P (2002) First isolation and characterization of Encephalitozoon cuniculi from a free-ranging rat (Rattus norvegicus). Vet Parasitol 107:279–285
Patterson-Kane JC, Caplazi P, Rurangirwa F, Tramontin RR, Wolfsdorf K (2003) Encephalitozoon cuniculi placentitis and abortion in a Quarterhorse mare. J Vet Diagn Invest 15:57–59
Reetz J, Rinder H, Thomschke A, Manke H, Schwebs M, Bruderek A (2002) First detection of the microsporidium Enterocytozoon bieneusi in non-mammalian hosts (chickens). Int J Parasitol 32:785–787
Rinder H, Thomschke A, Dengjel B, Gothe R, Loscher T, Zahler M (2000) Close genotypic relationship between Enterocytozoon bieneusi from humans and pigs and first detection in cattle. J Parasitol 86:185–188
Santin M, Trout JM, Fayer R (2004) Prevalence of Enterocytozoon bieneusi in post-weaned dairy calves in the eastern United States. Parasitol Res 93:287–289
Snowden KF, Shadduck JA (1999) Microsporidia in higher vertebrates. In Wittner M, Weiss LM (eds), The microsporidia and microsporidiosis. American Society for Microbiology, Washington, D.C.
Sparfel JM, Sarfati C, Liguory O, Caroff B, Dumoutier N, Gueglio B, Billaud E, Raffi F, Molina JM, 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 44:78S–78S
Sulaiman IM, Bern C, Gilman R, Cama V, Kawai V, Vargas D, Ticona E, Vivar A, Xiao LH (2003a) A molecular biologic study of Enterocytozoon bieneusi in HIV-infected patients in Lima, Peru. J Eukaryot Microbiol 50:591–596
Sulaiman IM, Fayer R, Lal AA, Trout JM, Schaefer FW, Xiao LH (2003b) Molecular characterization of microsporidia indicates that wild mammals harbor host-adapted Enterocytozoon spp as well as human-pathogenic Enterocytozoon bieneusi. Appl Environ Microbiol 69:4495–4501
Sulaliman IM, Fayer R, Yang CF, Santin M, Matos O, Xiao LH (2004) Molecular characterization of Enterocytozoon bieneusi in cattle indicates that only some isolates have zoonotic potential. Parasitol Res 92:328–334
Thurston-Enriquez JA, Watt P, Dowd SE, Enriquez J, Pepper IL, Gerba CP (2002) Detection of protozoan parasites and microsporidia in irrigation waters used for crop production. J Food Prot 65:378–382
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Kahler, A.M., Thurston-Enriquez, J.A. Human pathogenic microsporidia detection in agricultural samples: method development and assessment. Parasitol Res 100, 529–538 (2007). https://doi.org/10.1007/s00436-006-0300-2
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DOI: https://doi.org/10.1007/s00436-006-0300-2