Development of a New PCR Protocol to Detect and Subtype Blastocystis Spp
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Development of a new PCR protocol to detect and subtype Blastocystis spp. from humans and animals Mónica Santín, María Teresa Gómez- Muñoz, Gloria Solano-Aguilar & Ronald Fayer Parasitology Research Founded as Zeitschrift für Parasitenkunde ISSN 0932-0113 Volume 109 Number 1 Parasitol Res (2011) 109:205-212 DOI 10.1007/ s00436-010-2244-9 1 23 Your article is protected by copyright and all rights are held exclusively by Springer- Verlag (outside the USA). This e-offprint is for personal use only and shall not be self- archived in electronic repositories. If you wish to self-archive your work, please use the accepted author’s version for posting to your own website or your institution’s repository. You may further deposit the accepted author’s version on a funder’s repository at a funder’s request, provided it is not made publicly available until 12 months after publication. 1 23 Author's personal copy Parasitol Res (2011) 109:205–212 DOI 10.1007/s00436-010-2244-9 ORIGINAL PAPER Development of a new PCR protocol to detect and subtype Blastocystis spp. from humans and animals Mónica Santín & María Teresa Gómez-Muñoz & Gloria Solano-Aguilar & Ronald Fayer Received: 30 September 2010 /Accepted: 14 December 2010 /Published online: 6 January 2011 # Springer-Verlag (outside the USA) 2011 Abstract Blastocystis spp. is commonly found in the feces published primers and contains highly variable regions that of humans worldwide. Infection has been reported as allow phylogenetic analysis of Blastocystis. These primers asymptomatic, acute symptomatic, and chronic symptomatic. were used to detect and subtype Blastocystis spp. specimens This wide range of responses to infection could be related to from naturally infected humans, primates, cattle, pigs, and the genetic diversity of morphologically indistinguishable chickens. Based on these findings, application of this method specimens obtained from infected hosts. The former name can elucidate the complexity of this heterogeneous genus and Blastocystis hominis is now reported as Blastocystis spp. its role in human and animal disease, as well as its zoonotic because of its genetic diversity. Blastocystis is recognized as potential. 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 Introduction potential. Based on conserved regions of published nucleo- tide SSU rDNA sequences from all Blastocystis subtypes Blastocystis is one of the most common human intestinal found in GenBank, a PCR and sequencing protocol was parasites found in developed and developing countries (Tan developed. The ~500 bp SSU rDNA gene fragment 2004). This ubiquitous and enigmatic protozoan parasite amplified by this PCR is highly sensitive compared with has also been identified in a wide range of animals. The taxonomic status of Blastocystis has remained elusive until : recently, when small subunit ribosomal DNA (SSU rDNA) M. Santín (*) R. Fayer phylogeny (Silberman et al. 1996), as well as a combined Environmental Microbial and Food Safety Laboratory, analysis of eight molecules (Arisue et al. 2002) demon- Animal and Natural Resources Institute, Agricultural Research Service, United States Department of Agriculture, strated that it is a stramenopile. This eukaryotic group Building 173, BARC-East, 10300 Baltimore Avenue, includes unicellular and multicellular protists, including Beltsville, MD 20705, USA brown algae, diatoms, chrysophytes, water molds, and e-mail: [email protected] slime nets (Patterson 1994), and is a branch of the new M. T. Gómez-Muñoz higher level taxon Chromalveolata (Adl et al. 2005). Departamento de Sanidad Animal, Facultad de Veterinaria, Blastocystis is pleomorphic, presenting such a variety of Universidad Complutense de Madrid, forms, even within a monoculture, that identification of Avenida Puerta de Hierro S/N, specific stages is problematic (Tan 2008). The difficulty in 28040 Madrid, Spain identifying Blastocystis in fecal specimens has resulted in G. Solano-Aguilar confusion and misinterpretation regarding its life cycle, host United States Department of Agriculture, Beltsville Nutrition specificity, and pathogenicity. The pathogenic potential of Research Center, Diet Genomics and Immunology Laboratory, Blastocystis is controversial with numerous conflicting Agricultural Research Service, Building 307C, BARC-East, 10300 Baltimore Avenue, reports regarding its ability to cause disease (Vogelberg Beltsville, MD 20705, USA et al. 2010; Yakoob et al. 2010). Blastocystis has been found Author's personal copy 206 Parasitol Res (2011) 109:205–212 not only in individuals with gastrointestinal symptoms and fecal specimens containing Blastocystis were obtained from skin rash but also in apparently healthy and asymptomatic naturally infected hosts. One specimen was obtained from individuals (Boorom et al. 2008; Dominguez-Marquez et al. the American Type Culture Collection (ATCC # 50608D). 2009). Transmission of this parasite is also uncertain. It is All samples of animal origin, except for those from the generally accepted that transmission involves ingestion of chicken, were preliminarily identified as positive by micros- fecal matter following hand-to-hand, hand-to-food, or drink- copy. For all the human samples PCR was used to screen the ing contaminated water (Leelayoova et al. 2008; Stark et al. samples. Blastocystis in animal feces was concentrated by 2007; Tanizaki et al. 2005; Yoshikawa et al. 2000, 2004, CsCl centrifugation as previously described (Santín et al. 2009). Although specimens collected from infected humans 2004). Total DNA was extracted from each CsCl- and animals have been morphologically indistinguishable, concentrated fecal sample using a modification of the the application of molecular methods has shown remarkable DNeasy Tissue Kit (Qiagen, Valencia, CA). A 50-μl genetic diversity among specimens from both human and suspension of concentrated Blastocystis stages was sus- animals (Arisue et al. 2003;Noeletal.2003; Stensvold et al. pended in 180 μl of ATL buffer (supplied by the 2009a). Recently, a consensus terminology for subtypes (ST) manufacturer) and vortexed. To this suspension, 20 μlof of Blastocystis was developed on the basis of SSU-rDNA proteinase K (20 mg/ml) was added and the mixture was gene analysis and nine subtypes were established and incubated overnight at 55 C. The next day, 200 μlofAL designated as ST1 to ST9 (Stensvold et al. 2007). More buffer were added and purification proceeded as per recently, a tenth subtype (ST10) was described from both manufacturer's instructions. The nucleic acids were eluted primates and ungulates in Denmark (Stensvold et al. 2009a). in 100 μlofAEbuffer. It is likely that the confusion regarding Blastocystis Total DNA from human stools was extracted using the pathogenicity can be related to diagnostic limitations and QIAamp DNA stool minikit (Qiagen, Valencia, CA). differences in the virulence of different subtypes (Stensvold Briefly, 1 g of homogenized fecal sample was thawed, et al. 2009b, c). Blastocystis has low host specificity and is weighed, and resuspended with lysis buffer. The suspension considered a potential zoonotic pathogen, because infections was heated to 95 C to increase DNA yield, to remove in humans have been associated with contact with primates, inhibitors, and to increase proteinase K digestion before pigs, and poultry (Li et al. 2007;Noeletal.2005;Stensvold DNA was bound to a column, washed, and eluted in TE et al. 2009a, b; Yoshikawa et al. 2009). The subtype ST-2 buffer. was shared between local rhesus monkeys and children in Nepal, and therefore, monkeys were considered as a possible Gene amplification and sequencing source of Blastocystis ST-2 infection of humans (Yoshikawa et al. 2009). Similarly, Parkar et al. (2007) found ST-1 in To amplify a fragment of the SSU rDNA gene from various zoo-keepers and primates at the Perth Zoo. Pig ownership Blastocystis specimens, a PCR protocol was developed, was found to be a risk factor for Blastocystis in humans in using primers complementary to conserved regions of China (Li et al. 2007). In a study conducted in China, the published nucleotide SSU rDNA sequences of Blastocystis same subtype ST-5 was observed in 16 pigs, as well as in downloaded from GenBank (Table 2). A specific set of three humans living in the same rural area (Yan et al., 2007). primers were designed based upon multiple sequence The present study was undertaken to design, test, and alignment of the SSU rDNA gene. The primers forward select primers that target a region of the SSU rDNA gene Blast 505–532 (5′ GGA GGT AGT GAC AAT AAA TC 3′) that would facilitate detection and identification of subtypes (previously used by Böhm-Gloning et al. 1997) and reverse of Blastocystis in human and animal feces. The ultimate Blast 998–1017 (5′ TGC TTT CGC ACT TGT TCA TC 3′) goal is to use this new assay to increase our knowledge of amplifies a ca. 500 (479)-bp fragment, containing a variable subtypes regarding transmission routes, host specificity, region that allows subtyping of Blastocystis specimens. The zoonotic significance, and association with disease. locations of the primers based on reference nucleotide sequence U51151 are at nucleotide positions 445–464 and 905–924. Each 50-μl PCR mixture contained 1× PCR Material and methods buffer, 1.5 mM MgCl2, 0.2 mM dNTP, 2.5 U Taq (Qbiogene, Irvine, CA), 2.5 μl BSA (0.1 g/10 ml), and Sources of specimens, parasite purification,