Development of a new PCR protocol to detect and subtype 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

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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 . This eukaryotic group Building 173, BARC-East, 10300 Baltimore Avenue, includes unicellular and multicellular , including Beltsville, MD 20705, USA brown , , 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 (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, and DNA 1 μM of each primer. A total of 35 cycles, each consisting extraction of 95 C for 30 s, 54 C for 30 s, and 72 C for 30 s, was performed; an initial pre-heat step at 95 C for 4 min and Thirty-six Blastocystis specimens from humans and animals a final extension step at 72 C for 5 min were also were included in the present study (Table 1). Thirty-five included. Author's personal copy

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Table 1 Blastocystis isolates used in the study with information on hosts, locations, subtypes, and Genbank accession numbers

Isolate Host Location ST GenBank accession number

H-10 Human Colombia ST-1 HQ641595 H-14 Human Colombia ST-1 HQ641596 H-33 Human Colombia ST-1 HQ641597 H-42 Human Colombia ST-1 HQ641598 H-7 Human Colombia ST-2 (4 different nucleotide sequences) HQ641599-HQ641602 H-11 Human Colombia ST-2 (2 different nucleotide sequences) HQ641603- HQ641604 H-12 Human Colombia ST-2 HQ641605 H-29 Human Colombia ST-3 (5 different nucleotide sequences) HQ641606- HQ641610 H-30 Human Colombia ST-3 HQ641611 H-41 Human Colombia ST-3 HQ641612 H-57 Human Colombia ST-3 HQ641613 H-5 Human Colombia ST-1 (1 nucleotide sequence) HQ641614-HQ641620 ST-2 (6 nucleotide sequences) H-1 Human Spain ST-4 HQ641621 ATTC 50608D Human USA ST-4 HQ641622 C-3066 Cattle USA ST-10 HQ641623 C-3067 Cattle USA ST-10 HQ641624 C-3069 Cattle USA ST-10 HQ641625 C-3070 Cattle USA ST-10 HQ641626 C-3071 Cattle USA ST-10 HQ641627 C-3072 Cattle USA ST-10 HQ641628 C-3073 Cattle USA ST-10 HQ641629 P-5 Swine USA ST-5 HQ641630 P-6 Swine USA ST-5 HQ641631 P-8 Swine USA ST-5 HQ641632 P-12 Swine USA ST-5 HQ641633 P-13 Swine USA ST-5 HQ641634 P-20 Swine USA ST-5 HQ641635 P-18 C Swine Spain ST-5 HQ641636 PR-1 Primate (Hapalemur aureus) Spain ST-1 (5 nucleotide sequences) HQ641637-HQ641641 PR-4 Primate (Cercopithecus hamlyni) Spain ST-1 (1 nucleotide sequence) HQ641642-HQ641651 ST-2 (2 nucleotide sequences) ST-3 (7 nucleotide sequences) PR-5 Primate (Lemur catta) Spain ST-4 HQ641652 PR-9 Primate (Mandrillus leucophaeus) Spain ST-3 HQ641653 PR-11 Primate (Gorilla gorilla) Spain ST-2 (2 nucleotide sequences) HQ641654-HQ641655 PR-13 Primate (Cercocebus atys) Spain ST-3 HQ641656 PR-14 Primate (Cercocebus neglectus) Spain ST-3 HQ641657 Ch-1 Chicken Spain ST-6 (2 nucleotide sequences) HQ641658-HQ641661 ST-7 (2 nucleotide sequences)

PCR products were analyzed on 1% agarose gel, chemistries, and an ABI3100 sequencer analyzer (Applied visualized by ethidium bromide staining, and purified Biosystems, Foster City, CA). with Exonuclease I/Shrimp Alkaline Phosphatase (Exo- DNA from other organisms, SAP-IT™) (USB Corporation, Cleveland, Ohio). Purified Beltsville isolate, Giardia duodenalis Assemblage A isolate products were sequenced in both directions. The same WB, Giardia duodenalis Assemblage E isolate Beltsville, PCR primers were used in 10-μl reactions, Big Dye™ Enterocytozoon bieneusi genotype PtEb IX isolate 64, Author's personal copy

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Table 2 Reference sequences for each Blastocystis subtype ST GenBank Host Location Reference included in this study with their GenBank accession number, ST-1 U51151 Human USA Silberman et al. 1996 host, locations and reference AB107962 Human Japan Abe (2004) AB070993 Chicken Japan Arisue et al. (2003) AB070989 Human Japan Arisue et al. (2003) ST-2 AB070997 Primate Japan Arisue et al. (2003) AB107969 Primate Japan Abe (2004) AB070987 Human Japan Arisue et al. (2003) ST-3 AB091234 Human Japan Arisue et al. (2003) AB107963 Pig Japan Abe (2004) AB070992 Human Japan Arisue et al. (2003) ST-4 U26177 Guinea pig USA Leipe et al. (1996) AB071000 Rat Japan Arisue et al. (2003) AY244620 Human Germany Yoshikawa et al. (2004) ST-5 AB070998 Pig Japan Arisue et al. (2003) AB107964 Pig Japan Abe (2004) AB070999 Pig Japan Arisue et al. (2003) ST-6 AY135411 Turkey France Noel et al. (2003) AB107972 Bird Japan Abe (2004) AB070990 Human Japan Arisue et al. (2003) ST-7 AY590109 Human France Noel et al. (2003) AF408427 Human Japan Arisue et al. (2003) AB070996 Quail Japan Arisue et al. (2003) AY135409 Chicken France Noel et al. (2003) AB107973 Bird Japan Abe (2004) ST-8 AB107971 Bird Japan Abe (2004) AB107970 Primate Japan Abe (2004) ST-9 AF408425 Human Japan Yoshikawa et al. (2004) AF408426 Human Japan Yoshikawa et al. (2004) ST-10 FM164412 Cattle Denmark Stensvold et al. (Stensvold et al. 2009a, b, c) FM164413 Cattle Denmark Stensvold et al. (2009a, b, c)

Encephalitozoon hellem ATTC# 50451, Encephalitozoon sequenced in both directions using M13 forward and cuniculi ATTC# 50602, and Encephalitozoon intestinalis reverse primers. Up to ten clones from each specimen (DNA was extracted from spores originally isolated from an were sequenced. AIDS patient and grown in culture provided by Dr. Elizabeth Didier, Tulane Regional Primate Research Center, Phylogenetic analysis Covington, Louisiana), was also tested to assess the specificity of the primers. All sequences (excluding vector and primer sites) were The nucleotide sequences obtained in this study have subjected to BLAST searches in the GenBank database to been deposited in GenBank under accession numbers confirm specimens as Blastocystis spp. Subtype terminology HQ641595-HQ641661. for Blastocystis was used according to Stensvold et al. (2007). Nucleotide sequences for this study, as well as type Cloning sequences obtained from GenBank from each of the ten subtypes currently recognized, were aligned using the Clustal When mixed infection within a specimen was suspected W algorithm in the Megalign module (DNASTAR Inc., from the sequence traces, the PCR products of SSU Madison, WI). Clustal W determines that when a gap is rDNA were cloned using the TOPO TA cloning kit inserted, it can be removed only by editing, so final (Invitrogen Corp., Carlsbad, CA) and transformants were alignment adjustments were made manually to remove selected from each specimen and screened by PCR, and artificial gaps. Phylogenetic and molecular evolutionary Author's personal copy

Parasitol Res (2011) 109:205–212 209 analyses were made using MEGA software version 4 98.3%) was FM164413. These data confirm the identifi- (Tamura et al. 2007). Phylogenetic inference was by the cation of sequence specimens analyzed from cattle as neighbor-joining (NJ) method of Saitou and Nei (1987). ST-10. The phylogenetic tree revealed the presence of ten Genetic distance was calculated with the Kimura 2-parameter different clades that corresponded exactly to the ST 1 to model. Branch reliability was assessed using bootstrap 10, indicating that the sequence patterns of the variable analysis (1,000 replicates). regions amplified correlated well with the phylogenetic tree-based grouping. The sensitivity of the primers using this PCR protocol Results was determined by serial dilution of Blastocystis DNA. As little as 0.0001 ng of Blastocystis DNA (data not shown) A PCR amplicon of approximately 500 bp was successfully could be detected. The specificity of these primers was generated from all 36 specimens included in the study. All tested against samples of DNA known to be positive for C. PCR amplicons were sequenced to determine the subtypes parvum, G. duodenalis, E. bieneusi, E. hellem, E. cuniculi, following the subtype classification designated by Stensvold and E. intestinalis, and the PCR assay did not cross-react et al. (2007). Direct subtyping was accomplished for 28 with any of them (data not shown). specimens, detecting ST-1 in four human specimens, ST-2 in one human specimen, ST-3 in three humans and three primate specimens, ST-4 in two human and one primate Discussion specimens, ST-5 in seven pig specimens, and ST-10 in seven cattle specimens (Table 1). However, in eight specimens Blastocystis is a common intestinal parasite of humans and (H-5, H-7, H-11, H-29, PR-1, PR-4, PR-11, and Ch-1), animals. Diagnosis is difficult, relying on light microscopy mixed infection within a specimen were suspected from of fecal smears, which has low sensitivity; or on culture the sequence traces, and those samples were subjected to methods that are more sensitive, but time-consuming and cloning. Heterogeneity in nucleotide sequences were not available in most diagnostic laboratories. In contrast, observed among clones (Table 1). In five specimens, three PCR has been found to be a rapid and highly sensitive tool human (H-7, H-11, and H-29) and two primate (PR-1 and for the identification of many parasites, essential for PR-11) all sequences for each single specimen were located detecting genetic variation between organisms that are within an independent clade (ST-1, ST-2, or ST-3). The morphologically indistinguishable, such as genotypes of heterogeneous sequences from one primate specimen (PR-1) Giardia and species of Cryptosporidium. Several methods, were all located within ST-1; from three specimens, two including arbitrarily primed PCR and subtype-specific human (H-7 and H-11), and one primate (PR-11), within ST- sequence-tagged site (STS) primers, have been developed 2 clade; and from one human specimen (H-29) within ST-3 and used in studies to detect genetic variations among clade. However, in three specimens, one human (H-5), Blastocystis specimens (Arisue et al. 2003; Yoshikawa et al. one primate (PR-4), and one chicken (Ch-1), nucleotide 2003). In the present study, a pair of PCR primers was sequences were located in different ST clades. Human successfully developed for the identification and subtyping specimen H-5 nucleotide sequences were located in two ST using direct sequencing of Blastocystis. The specificity of clades, ST-1, and ST-2; primate specimen PR-4 nucleotide these primers was confirmed by the successful amplifica- sequences were located in three, ST-1, ST-2, and ST-3; and tion of DNA from all Blastocystis specimens included in the chicken specimen CH-1 nucleotide sequences were the study and their inability to amplify DNA from other located in two clades ST-6 and ST-7. parasites. The assay developed in this study was A phylogenetic analysis of all sequences obtained in compared with previous published protocols that used PCR this study was carried out together with reference for subtyping of Blastocystis. Our PCR protocol has the sequences (Table 2) obtained from GenBank (Fig. 1). advantage of greater sensitivity than the PCR method used The two reference sequences from subtype 10 (ST-10) by Stensvold et al. (2006, 2009a, b, c). PCRs described by were excluded from the construction of the phylogenetic Stensvold et al. (2006) and (2009a) were able to detect tree, since the overlapping region with sequences from our 0.01 ng and 0.001 ng of Blastocystis DNA, respectively. study was not long enough for FM164413, and sequence PCR by Parkar et al. (2007) has the same sensitivity as our FM164412 did not overlap with our nucleotide sequence. PCR, but our PCR is a one-step PCR, whereas the PCR However, when a BLAST search was conducted including used by Parkar et al. is a nested PCR. The possibility of only the area that overlaps among our cattle nucleotide contamination increases when using a nested PCR. The sequences and FM164413 (240 bp that includes nucleo- PCR method used by Menounos et al. (2008) is more tides from position 365 to 604 from reference nucleotide sensitive than our PCR; they were able to detect 0.00001 ng sequence FM164413), the closest match (similarity of of Blastocystis DNA. However, the disadvantage of using Author's personal copy

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Fig. 1 Phylogenetic tree of 51 H-5.4 Colombia (ST-2) H-12 Colombia (ST-2) the SSU-rDNA gene sequences H-11.1 Colombia (ST-2) of Blastocystis specimens was H-5.7 Colombia (ST-2) inferred using the neighbor- 64 H-7.1 Colombia (ST-2) 70 PR-4.2 Spain (ST-2) joining method. Reference ST-2 Human Japan (AB070987) sequences from GenBank 93 PR-4.3 Spain (ST-2) ST-2 Primate Japan (AB070997) have the accession number PR-11.1 Spain (ST-2) 100 in parenthesis. Bootstrap ST-2 Primate Japan (AB107969) PR-11.2 Spain (ST-2) proportions (%) are attached H-11.2 Colombia (ST-2) to the internal branches (1,000 99 H-5.1 Colombia (ST-2) H-5.6 Colombia (ST-2) 92 replicates). Names of the H-7.2 Colombia (ST-2) specimens, host, and locations, H-7.4 Colombia (ST-2) 56 H-5.2 Colombia (ST-2) 100 as well as the GenBank acces- H-5.5 Colombia (ST-2) sion numbers are shown in H-7.3 Colombia (ST-2) parenthesis. Bootstrap values ST-1 Human Japan (AB107962) 68 H-10 Colombia (ST-1) of less than 50% are 95 ST-1 Human Japan (AB070989) not shown H-14 Colombia (ST-1) ST-1 Human Japan (U51151) 97 PR-1.1 Spain (ST-1) 77 PR-1.2 Spain (ST-1) 100 PR-4.1 Spain (ST-1) 74 ST-1 Chicken Japan (AB070993) 66 H-42 Colombia (ST-1) 83 PR-1.4 Spain (ST-1) PR-1.5 Spain (ST-1)

95 H-33 Colombia (ST-1) H-5.3 Colombia (ST-1) 59 PR-1.3 Spain (ST-1) 73 ST-5 Pig Japan (AB107964) 69 P-18C Spain (ST-5) 67 ST-5 Pig Japan (AB070998) ST-5 Pig Japan (AB070999) 69 P-5 USA (ST-5) 100 P-6 USA (ST-5) P-12 USA (ST-5) 96 P-13 USA (ST-5) P-8 USA (ST-5) 65 P-20 USA (ST-5) PR-4.5 Spain (ST-3) PR-4.7 Spain (ST-3) 63 PR-4.8 Spain (ST-3) PR-4.6 Spain (ST-3) 100 PR-14 Spain (ST 3) 99 53 PR-4.9 Spain (ST-3) ST-3 Human Japan (AB070992) PR-4.10 Spain (ST-3) 99 PR-9 Spain (ST-3) 92 PR-4.4 Spain (ST-3) PR-13 Spain (ST-3) H-29.4 Colombia (ST-3) 70 H-29.5 Colombia (ST-3)

90 H-41 Colombia (ST-3) H-29.3 Colombia (ST-3) 67 ST-3 Pig Japan (AB107963) H-29.1 Colombia (ST-3) ST-3 Human Japan (AB091234) H-57 Colombia (ST-3) H-29.2 Colombia (ST-3) 60 H-30 Colombia (ST-3) 100 ST-9 Human Japan (AF408425) ST-9 Human Japan (AF408426)

100 ST-6 Bird Japan (AB107972) ST-6 Human Japan (AB070990) 100 Ch-1.2 Spain (ST-6) 89 Ch-1.1 Spain (ST-6) 79 89 ST-6 Turkey Japan (AY135411) C-3067 USA (ST-10) C-3069 USA (ST-10) C-3071 USA (ST-10) 100 C-3073 USA (ST-10) C-3066 USA (ST-10) C-3070 USA (ST-10) C-3072 USA (ST-10) 91 100 ST-8 Primate Japan (AB107970) ST-8 Bird Japan (AB107971) PR-5 Spain (ST-4) 98 64 ST-4 Guinea pig USA (U26177) 99 ST-4 Rat Japan (AB071000)

52 H-1 Spain (ST-4) ST-4 Human Germany (AY244620) 52 50 ATTC 50608D USA (ST-4) ST-7 Human France (AY135409) 99 ST-7 Bird Japan (AB107973) 100 Ch-1.4 Spain (ST-7) 99 ST-7 Human France (AY590109) 99 Ch-1.3 Spain (ST-7) 99 ST-7 Human Japan (AF408427) 87 ST-7 Quail Japan (AB070996)

0.02 Author's personal copy

Parasitol Res (2011) 109:205–212 211 the PCR method of Menounos et al. is that the amplicon ST-7), and three subtypes were present in PR-4 (ST-1, ST-2, amplified by their PCR method is very short, only 260 bp. and ST-3). Although this would not be a problem for diagnostic Pathogenicity of Blastocystis is a controversial matter purposes, it clearly limits the accuracy required for and additional research is needed to determine potential subtyping. differences in pathogenicity of Blastocystis infections Blastocystis has been found not only in humans but also associated with different subtypes. Application of the in a wide range of animals, such as nonhuman primates, method developed and tested in the present study can pigs, cattle, rodents, and birds (Abe 2004; Arisue et al. elucidate the complexity of this heterogeneous genus and 2003; Navarro et al. 2008; Noel et al. 2005; Stensvold et al. its role in human or animal diseases, as well as its zoonotic 2009a). The mode of transmission is unclear but may be potential. associated with animal contact and with the ingestion of food or water contaminated with cysts from reservoir hosts Acknowledgment The authors thank Meghan Heffron for her (Al-Binali et al. 2006; Leelayoova et al. 2004, 2008; Parkar technical services in support of this study. et al. 2007; Salim et al. 1999; Suresh et al. 2005; Taamasri et al. 2000). Blastocystis displays a broad genetic diversity References and probably comprises many species based on the evolutionary distance observed among different subtypes, which is comparable to or greater than that observed among Abe N (2004) Molecular and phylogenetic analysis of Blastocystis – other stramenopile species (Noel et al. 2005). However, at isolates from various hosts. Vet Parasitol 120:235 242 Adl S, Simpson A, Farmer M, Andersen R, Anderson O, Barta J, present, there is not enough information within the genus Bowser S, Brugerolle G, Fensome R, Fredericq S, James T, Blastocystis except to indicate that ten subtypes exist. Karpov S, Kugrens P, Krug J, Lane C, Lewis L, Lodge J, Lynn Blastocystis has low host specificity, and previous studies D, Mann D, McCourt R, Mendoza L, Moestrup O, Mozley- suggested its zoonotic potential. Some specimens from Standridge S, Nerad T, Shearer C, Smirnov A, Spiegel F, Taylor M (2005) The new higher level classification of with animal origin have been regarded as zoonotic, based on emphasis on the of protists. J Eukaryot Microbiol genotypic homology to human specimens (Arisue et al. 52:399–451 2003; Noel et al. 2003, 2005). Primers developed in the Al-Binali AM, Bello CS, El-Shewy K, Abdulla SE (2006) The present study were able to detect and subtype using direct prevalence of parasites in commonly used leafy vegetables in South Western, Saudi Arabia. Saudi Med J 27:613–616 sequencing Blastocystis spp. specimens from naturally Arisue N, Hashimoto T, Yoshikawa H, Nakamura Y, Nakamura G, infected cattle, pigs, humans, primates, and chickens, Nakamura F, Yano T, Hasegawa M (2002) Phylogenetic position identifying eight of the ten subtypes currently recognized, of Blastocystis hominis and of inferred from multiple – and thereby, establishing its usefulness for the identification molecular sequence data. J Eukaryot Microbiol 49:42 53 Arisue N, Hashimoto T, Yoshikawa H (2003) Sequence heterogeneity and subtyping of Blastocystis specimens collected from of the small subunit ribosomal RNA genes among Blastocystis humans and animals. isolates. Parasitology 126:1–9 The subtype distribution detected in this study reflects Böhm-Gloning B, Knobloch J, Walderich B (1997) Five subgroups of the subtype distribution in different host reported in Blastocystis hominis from symptomatic and asymptomatic patients revealed by restriction site analysis of PCR-amplified previous reports (Stensvold et al. 2009a). All specimens 16S-like rDNA. Trop Med Int Health 2:771–778 identified as ST-1, ST-2, ST-3, and ST-4 in the present Boorom KF, Smith H, Nimri L, Viscogliosi E, Spanakos G, Parkar U, study were from human and primate specimens, whereas all Li LH, Zhou XN, Ok UZ, Leelayoova S, Jones MS (2008) Oh specimens from pigs were ST-5, and all cattle specimens my aching gut: , Blastocystis, and asymptomatic infection. Parasit Vectors 1:40 were ST-10. Although specimen PR-4.5 was identified as Dominguez-Marquez MV, Guna R, Munoz C, Gomez-Munoz MT, ST-4 in the phylogenetic tree, it could represent a new Borras R (2009) High prevalence of subtype 4 among isolates of clade. The chicken specimen examined in this study was a Blastocystis hominis from symptomatic patients of a health – mixed infection with subtypes ST-6 and ST-7, both of district of Valencia (Spain). Parasitol Res 105:949 955 Leelayoova S, Rangsin R, Taamasri P, Naaglor T, Thathaisong U, which ST are known to contain mostly bird specimens Mungthin M (2004) Evidence of waterborne transmission of (Stensvold et al. 2009a). Sequence heterogeneity was found Blastocystis hominis. Amer J Trop Med Hyg 70:658–662 among different clones in eight specimens (H-5, H-7, H-11, Leelayoova S, Siripattanapipong S, Thathaisong U, Naaglor T, H-29, PR-1, PR-4, PR-11, and Ch-1). It is possible that the Taamasri P, Piyaraj P, Mungthin M (2008) Drinking water: a possible source of Blastocystis spp. subtype 1 infection in heterogeneity is due to the presence of multi-copy genes of schoolchildren of a rural community in central Thailand. Amer SSU rDNA in the Blastocystis genome. However, clear J Trop Med Hyg 79:401–406 mixed infections with more than one ST were present in Leipe DD, Tong SM, Goggin CL, Slemenda SB, Pieniazek NJ, three specimens: human specimen H-5, primate specimen Sogin ML (1996) 16S-like rDNA sequences from Developayella elegans, Labyrinthuloides haliotidis, and lacertae PR-4, and chicken specimen Ch-1. Two subtypes were confirm that the stramenopiles are a primarily heterotrophic present in H-5 (ST-1 and ST-2) and in Ch-1 (ST-6 and group. Europ. J Protistol 32: 449-458 Author's personal copy

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