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Parasitology Research

, Volume 109, Issue 1, pp 205–212 | Cite as

Development of a new PCR protocol to detect and subtype Blastocystis spp. from humans and animals

  • Mónica SantínEmail author
  • María Teresa Gómez-Muñoz
  • Gloria Solano-Aguilar
  • Ronald Fayer
Original Paper

Abstract

Blastocystis spp. is commonly found in the feces of humans worldwide. Infection has been reported as asymptomatic, acute symptomatic, and chronic symptomatic. This wide range of responses to infection could be related to the genetic diversity of morphologically indistinguishable specimens obtained from infected hosts. The former name Blastocystis hominis is now reported as Blastocystis spp. because of its genetic diversity. Blastocystis is recognized as a complex of subtypes that have not been fully characterized as independent species. The finding of Blastocystis spp. in feces from several animal species suggests a zoonotic potential. Based on conserved regions of published nucleotide SSU rDNA sequences from all Blastocystis subtypes found in GenBank, a PCR and sequencing protocol was developed. The ~500 bp SSU rDNA gene fragment amplified by this PCR is highly sensitive compared with published primers and contains highly variable regions that allow phylogenetic analysis of Blastocystis. These primers were used to detect and subtype Blastocystis spp. specimens from naturally infected humans, primates, cattle, pigs, and chickens. Based on these findings, application of this method can elucidate the complexity of this heterogeneous genus and its role in human and animal disease, as well as its zoonotic potential.

Keywords

Human Specimen Zoonotic Potential Blastocystis Infection Primate Specimen Enterocytozoon Bieneusi 
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

Acknowledgment

The authors thank Meghan Heffron for her technical services in support of this study.

References

  1. Abe N (2004) Molecular and phylogenetic analysis of Blastocystis isolates from various hosts. Vet Parasitol 120:235–242PubMedCrossRefGoogle Scholar
  2. Adl S, Simpson A, Farmer M, Andersen R, Anderson O, Barta J, Bowser S, Brugerolle G, Fensome R, Fredericq S, James T, Karpov S, Kugrens P, Krug J, Lane C, Lewis L, Lodge J, Lynn D, Mann D, McCourt R, Mendoza L, Moestrup O, Mozley-Standridge S, Nerad T, Shearer C, Smirnov A, Spiegel F, Taylor M (2005) The new higher level classification of eukaryotes with emphasis on the taxonomy of protists. J Eukaryot Microbiol 52:399–451PubMedCrossRefGoogle Scholar
  3. Al-Binali AM, Bello CS, El-Shewy K, Abdulla SE (2006) The prevalence of parasites in commonly used leafy vegetables in South Western, Saudi Arabia. Saudi Med J 27:613–616PubMedGoogle Scholar
  4. Arisue N, Hashimoto T, Yoshikawa H, Nakamura Y, Nakamura G, Nakamura F, Yano T, Hasegawa M (2002) Phylogenetic position of Blastocystis hominis and of stramenopiles inferred from multiple molecular sequence data. J Eukaryot Microbiol 49:42–53PubMedCrossRefGoogle Scholar
  5. Arisue N, Hashimoto T, Yoshikawa H (2003) Sequence heterogeneity of the small subunit ribosomal RNA genes among Blastocystis isolates. Parasitology 126:1–9PubMedCrossRefGoogle Scholar
  6. Böhm-Gloning B, Knobloch J, Walderich B (1997) Five subgroups of Blastocystis hominis from symptomatic and asymptomatic patients revealed by restriction site analysis of PCR-amplified 16S-like rDNA. Trop Med Int Health 2:771–778PubMedCrossRefGoogle Scholar
  7. Boorom KF, Smith H, Nimri L, Viscogliosi E, Spanakos G, Parkar U, Li LH, Zhou XN, Ok UZ, Leelayoova S, Jones MS (2008) Oh my aching gut: irritable bowel syndrome, Blastocystis, and asymptomatic infection. Parasit Vectors 1:40PubMedCrossRefGoogle Scholar
  8. Dominguez-Marquez MV, Guna R, Munoz C, Gomez-Munoz MT, Borras R (2009) High prevalence of subtype 4 among isolates of Blastocystis hominis from symptomatic patients of a health district of Valencia (Spain). Parasitol Res 105:949–955PubMedCrossRefGoogle Scholar
  9. Leelayoova S, Rangsin R, Taamasri P, Naaglor T, Thathaisong U, Mungthin M (2004) Evidence of waterborne transmission of Blastocystis hominis. Amer J Trop Med Hyg 70:658–662Google Scholar
  10. Leelayoova S, Siripattanapipong S, Thathaisong U, Naaglor T, Taamasri P, Piyaraj P, Mungthin M (2008) Drinking water: a possible source of Blastocystis spp. subtype 1 infection in schoolchildren of a rural community in central Thailand. Amer J Trop Med Hyg 79:401–406Google Scholar
  11. Leipe DD, Tong SM, Goggin CL, Slemenda SB, Pieniazek NJ, Sogin ML (1996) 16S-like rDNA sequences from Developayella elegans, Labyrinthuloides haliotidis, and Proteromonas lacertae confirm that the stramenopiles are a primarily heterotrophic group. Europ. J Protistol 32: 449-458Google Scholar
  12. Li LH, Zhou XN, Du ZW, Wang XZ, Wang LB, Jiang JY, Yoshikawa H, Steinmann P, Utzinger J, Wu Z, Chen JX, Chen SH, Zhang L (2007) Molecular epidemiology of human Blastocystis in a village in Yunnan province, China. Parasitol Int 56:281–286PubMedCrossRefGoogle Scholar
  13. Menounos PG, Spanakos G, Tegos N, Vassalos CM, Papadopoulou C, Vakalis NC (2008) Direct detection of Blastocystis sp. in human faecal samples and subtype assignment using single strand conformational polymorphism and sequencing. Mol Cell Probes 22:24–29PubMedCrossRefGoogle Scholar
  14. Navarro C, Dominguez-Marquez MV, Garijo-Toledo MM, Vega-Garcia S, Fernandez-Barredo S, Perez-Gracia MT, Garcia A, Borras R, Gomez-Munoz MT (2008) High prevalence of Blastocystis sp. in pigs reared under intensive growing systems: frequency of ribotypes and associated risk factors. Vet Parasitol 153:347–358PubMedCrossRefGoogle Scholar
  15. Noel C, Peyronnet C, Gerbod D, Edgcomb VP, Delgado-Viscogliosi P, Sogin ML, Capron M, Viscogliosi E, Zenner L (2003) Phylogenetic analysis of Blastocystis isolates from different hosts based on the comparison of small-subunit rRNA gene sequences. Mol Biochem Parasitol 126:119–123PubMedCrossRefGoogle Scholar
  16. Noel C, Dufernez F, Gerbod D, Edgcomb VP, Delgado-Viscogliosi P, Ho L, Singh M, Wintjens R, Sogin ML, Capron M, Pierce R, Zenner L, Viscogliosi E (2005) Molecular phylogenies of Blastocystis isolates from different hosts: implications for genetic diversity, identification of species, and zoonosis. J Clin Microbiol 43:348–355PubMedCrossRefGoogle Scholar
  17. Parkar U, Traub RJ, Kumar S, Mungthin M, Vitali S, Leelayoova S, Morris K, Thompson RC (2007) Direct characterization of Blastocystis from faeces by PCR and evidence of zoonotic potential. Parasitology 134:359–367PubMedCrossRefGoogle Scholar
  18. Patterson DJ (1994) Protozoa, evolution and systematics, In: Hausmann K, Hülsmann N (eds) Progress in protozoology, Proc IX Internat Cong Protozool. Stuttgart: Fischer, Stuttgart, pp 1–14Google Scholar
  19. Salim HR, Kumar GS, Vellayan S, Mak JW, Anuar AK, Vennila GD, Saminathan R, Ramakrishnan K (1999) Blastocystis in animal handlers. Parasitol Res 85:1032–1033CrossRefGoogle Scholar
  20. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425PubMedGoogle Scholar
  21. Santín M, Trout JM, Xiao L, Zhou L, Greiner E, Fayer R (2004) Prevalence and age-related variation of Cryptosporidium species and genotypes in dairy calves. Vet Parasitol 122:103-117Google Scholar
  22. Silberman JD, Sogin ML, Leipe DD, Clark CG (1996) Human parasite finds taxonomic home. Nature 380:398PubMedCrossRefGoogle Scholar
  23. Stark D, Fotedar R, van Hal S, Beebe N, Marriott D, Ellis JT, Harkness J (2007) Prevalence of enteric protozoa in human immunodeficiency virus (HIV)-positive and HIV-negative men who have sex with men from Sydney, Australia. Am J Trop Med Hyg 76:549–552PubMedGoogle Scholar
  24. Stensvold CR, Suresh GK, Tan KSW, Thompson RCA, Traub R, Viscogliosi E, Yoshikawa H, Clark CG (2007) Terminology for Blastocystis subtypes–a consensus. Trends Parasitol 23:93–96PubMedCrossRefGoogle Scholar
  25. Stensvold CR, Alfellani MA, Norskov-Lauritsen S, Prip K, Victory EL, Maddox C, Nielsen HV, Clark CG (2009a) Subtype distribution of Blastocystis isolates from synanthropic and zoo animals and identification of a new subtype. Int J Parasitol 39:473–479PubMedCrossRefGoogle Scholar
  26. Stensvold CR, Lewis HC, Hammerum AM, Porsbo LJ, Nielsen SS, Olsen KE, Arendrup MC, Nielsen HV, Molbak K (2009b) Blastocystis: unravelling potential risk factors and clinical significance of a common but neglected parasite. Epidemiol Infect 137:1655–1663PubMedCrossRefGoogle Scholar
  27. Stensvold CR, Nielsen HV, Molbak K, Smith HV (2009c) Pursuing the clinical significance of Blastocystis–diagnostic limitations. Trends Parasitol 25:23–29PubMedCrossRefGoogle Scholar
  28. Stesvold R, Brillowska-Dabrowska A, Nielsen HV, Arendrup MC (2006) Detection of Blastocystis hominis in unpreserved stool specimens by using polymerase chain reaction. J Parasitol 95:1081–1087CrossRefGoogle Scholar
  29. Suresh K, Smith HV, Tan TC (2005) Viable Blastocystis cysts in Scottish and Malaysian sewage samples. Appl Environ Microbiol 71:5619–5620PubMedCrossRefGoogle Scholar
  30. Taamasri P, Mungthin M, Rangsin R, Tongupprakarn B, Areekul W, Leelayoova S (2000) Transmission of intestinal blastocystosis related to the quality of drinking water. Southeast Asian J Trop Med Public Health 31:112–117PubMedGoogle Scholar
  31. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molec Biol Evolut 24:1596–1599CrossRefGoogle Scholar
  32. Tan KS (2004) Blastocystis in humans and animals: new insights using modern methodologies. Vet Parasitol 126:121–144PubMedCrossRefGoogle Scholar
  33. Tan KS (2008) New insights on classification, identification, and clinical relevance of Blastocystis spp. Clin Microbiol Rev 21:639–665PubMedCrossRefGoogle Scholar
  34. Tanizaki A, Yoshikawa H, Iwatani S, Kimata I (2005) Infectivity of Blastocystis isolates from chickens, quails and geese in chickens. Parasitol Res 96:57–61PubMedCrossRefGoogle Scholar
  35. Vogelberg C, Stensvold CR, Monecke S, Ditzen A, Stopsack K, Heinrich-Grafe U, Pohlmann C (2010) Blastocystis sp. subtype 2 detection during recurrence of gastrointestinal and urticarial symptoms. Parasitol Int 59:469–471PubMedCrossRefGoogle Scholar
  36. Yakoob J, Jafri W, Beg MA, Abbas Z, Naz S, Islam M, Khan R (2010) Irritable bowel syndrome: is it associated with genotypes of Blastocystis hominis. Parasitol Res 106:1033–1038PubMedCrossRefGoogle Scholar
  37. Yan Y, Su S, Ye J, Lai X, Lai R, Liao H, Chen G, Zhang R, Hou Z, Luo X (2007) Blastocystis sp. subtype 5: a possibly zoonotic genotype. Parasitol Res 101:1527–1532PubMedCrossRefGoogle Scholar
  38. Yoshikawa H, Abe N, Iwasawa M, Kitano S, Nagano I, Wu Z, Takahashi Y (2000) Genomic analysis of Blastocystis hominis strains isolated from two long-term health care facilities. J Clin Microbiol 38:1324–1330PubMedGoogle Scholar
  39. Yoshikawa H, Abe N, Wu Z (2003) Genomic polymorphism among Blastocystis isolates and development of PCR-based identification of zoonotic isolates. J Eukaryot Microbiol 50:710–711PubMedCrossRefGoogle Scholar
  40. Yoshikawa H, Yoshida K, Nakajima A, Yamanari K, Iwatani S, Kimata I (2004) Fecal-oral transmission of the cyst form of Blastocystis hominis in rats. Parasitol Res 94:391–396PubMedCrossRefGoogle Scholar
  41. Yoshikawa H, Wu Z, Pandey K, Pandey BD, Sherchand JB, Yanagi T, Kanbara H (2009) Molecular characterization of Blastocystis isolates from children and rhesus monkeys in Katmandu, Nepal. Vet Parasitol 160:295–300PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag (outside the USA) 2011

Authors and Affiliations

  • Mónica Santín
    • 1
    Email author
  • María Teresa Gómez-Muñoz
    • 2
  • Gloria Solano-Aguilar
    • 3
  • Ronald Fayer
    • 1
  1. 1.Environmental Microbial and Food Safety LaboratoryAnimal and Natural Resources Institute, Agricultural Research Service, United States Department of AgricultureBeltsvilleUSA
  2. 2.Departamento de Sanidad Animal, Facultad de VeterinariaUniversidad Complutense de MadridMadridSpain
  3. 3.United States Department of Agriculture, Beltsville Nutrition Research CenterDiet Genomics and Immunology Laboratory, Agricultural Research ServiceBeltsvilleUSA

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