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

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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.

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References

  • Abe N (2004) Molecular and phylogenetic analysis of Blastocystis isolates from various hosts. Vet Parasitol 120:235–242

    Article  PubMed  CAS  Google Scholar 

  • 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 52:399–451

    Article  PubMed  Google Scholar 

  • 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 27:613–616

    PubMed  Google Scholar 

  • 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 49:42–53

    Article  PubMed  CAS  Google Scholar 

  • Arisue N, Hashimoto T, Yoshikawa H (2003) Sequence heterogeneity of the small subunit ribosomal RNA genes among Blastocystis isolates. Parasitology 126:1–9

    Article  PubMed  CAS  Google Scholar 

  • 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 2:771–778

    Article  PubMed  Google Scholar 

  • 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 1:40

    Article  PubMed  Google Scholar 

  • 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 105:949–955

    Article  PubMed  Google Scholar 

  • 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 70:658–662

    Google Scholar 

  • 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 79:401–406

    CAS  Google Scholar 

  • 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: 449-458

    Google Scholar 

  • 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 56:281–286

    Article  PubMed  CAS  Google Scholar 

  • 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 22:24–29

    Article  PubMed  CAS  Google Scholar 

  • 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 153:347–358

    Article  PubMed  CAS  Google Scholar 

  • 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 126:119–123

    Article  PubMed  CAS  Google Scholar 

  • 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 43:348–355

    Article  PubMed  CAS  Google Scholar 

  • 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 134:359–367

    Article  PubMed  CAS  Google Scholar 

  • 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 1–14

  • Salim HR, Kumar GS, Vellayan S, Mak JW, Anuar AK, Vennila GD, Saminathan R, Ramakrishnan K (1999) Blastocystis in animal handlers. Parasitol Res 85:1032–1033

    Article  Google Scholar 

  • Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425

    PubMed  CAS  Google Scholar 

  • 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 122:103-117

    Google Scholar 

  • Silberman JD, Sogin ML, Leipe DD, Clark CG (1996) Human parasite finds taxonomic home. Nature 380:398

    Article  PubMed  CAS  Google Scholar 

  • 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 76:549–552

    PubMed  Google Scholar 

  • 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 23:93–96

    Article  PubMed  Google Scholar 

  • 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 39:473–479

    Article  PubMed  CAS  Google Scholar 

  • 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 137:1655–1663

    Article  PubMed  CAS  Google Scholar 

  • Stensvold CR, Nielsen HV, Molbak K, Smith HV (2009c) Pursuing the clinical significance of Blastocystis–diagnostic limitations. Trends Parasitol 25:23–29

    Article  PubMed  Google Scholar 

  • 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 95:1081–1087

    Article  Google Scholar 

  • Suresh K, Smith HV, Tan TC (2005) Viable Blastocystis cysts in Scottish and Malaysian sewage samples. Appl Environ Microbiol 71:5619–5620

    Article  PubMed  CAS  Google Scholar 

  • 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 31:112–117

    PubMed  CAS  Google Scholar 

  • Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molec Biol Evolut 24:1596–1599

    Article  CAS  Google Scholar 

  • Tan KS (2004) Blastocystis in humans and animals: new insights using modern methodologies. Vet Parasitol 126:121–144

    Article  PubMed  Google Scholar 

  • Tan KS (2008) New insights on classification, identification, and clinical relevance of Blastocystis spp. Clin Microbiol Rev 21:639–665

    Article  PubMed  CAS  Google Scholar 

  • Tanizaki A, Yoshikawa H, Iwatani S, Kimata I (2005) Infectivity of Blastocystis isolates from chickens, quails and geese in chickens. Parasitol Res 96:57–61

    Article  PubMed  Google Scholar 

  • 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 59:469–471

    Article  PubMed  Google Scholar 

  • 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 106:1033–1038

    Article  PubMed  Google Scholar 

  • 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 101:1527–1532

    Article  PubMed  Google Scholar 

  • 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 38:1324–1330

    PubMed  CAS  Google Scholar 

  • Yoshikawa H, Abe N, Wu Z (2003) Genomic polymorphism among Blastocystis isolates and development of PCR-based identification of zoonotic isolates. J Eukaryot Microbiol 50:710–711

    Article  PubMed  Google Scholar 

  • 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 94:391–396

    Article  PubMed  Google Scholar 

  • 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 160:295–300

    Article  PubMed  CAS  Google Scholar 

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Acknowledgment

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

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Authors and Affiliations

  1. Environmental Microbial and Food Safety Laboratory, Animal and Natural Resources Institute, Agricultural Research Service, United States Department of Agriculture, Building 173, BARC-East, 10300 Baltimore Avenue, Beltsville, MD, 20705, USA

    Mónica Santín & Ronald Fayer

  2. Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Avenida Puerta de Hierro S/N, 28040, Madrid, Spain

    María Teresa Gómez-Muñoz

  3. United States Department of Agriculture, Beltsville Nutrition Research Center, Diet Genomics and Immunology Laboratory, Agricultural Research Service, Building 307C, BARC-East, 10300 Baltimore Avenue, Beltsville, MD, 20705, USA

    Gloria Solano-Aguilar

Authors
  1. Mónica Santín
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  2. María Teresa Gómez-Muñoz
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  3. Gloria Solano-Aguilar
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  4. Ronald Fayer
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Corresponding author

Correspondence to Mónica Santín.

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Santín, M., Gómez-Muñoz, M.T., Solano-Aguilar, G. et al. Development of a new PCR protocol to detect and subtype Blastocystis spp. from humans and animals. Parasitol Res 109, 205–212 (2011). https://doi.org/10.1007/s00436-010-2244-9

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  • DOI: https://doi.org/10.1007/s00436-010-2244-9

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