This article was originally published in a journal published byElsevier, and the attached copy is provided by Elsevier for the

author’s benefit and for the benefit of the author’s institution, fornon-commercial research and educational use including without

limitation use in instruction at your institution, sending it to specificcolleagues that you know, and providing a copy to your institution’s

administrator.

All other uses, reproduction and distribution, including withoutlimitation commercial reprints, selling or licensing copies or access,

or posting on open internet sites, your personal or institution’swebsite or repository, are prohibited. For exceptions, permission

may be sought for such use through Elsevier’s permissions site at:

http://www.elsevier.com/locate/permissionusematerial

Autho

r's p

erso

nal

copy

Short communication

Genetic diversity of glucose phosphate isomerase fromEntamoeba histolytica!

Elham Razmjou a, Ali Haghighi b, Mostafa Rezaian a, Seiki Kobayashi c, Tomoyoshi Nozaki d,!

a Department of Medical Parasitology and Mycology, School of Public Health and Institute of Public Health Research, Tehran University of Medical Sciences, Iranb Department of Parasitology and Mycology, Shaheed Beheshti University of Medical Sciences, Iranc Department of Tropical Medicine and Parasitology, School of Medicine, Keio University, Japan

d Department of Parasitology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan

Received 28 June 2006; received in revised form 30 July 2006; accepted 3 August 2006Available online 18 September 2006

Abstract

To investigate the molecular basis of zymodeme analysis in the enteric protozoan parasite Entamoeba histolytica, genes encoding glucosephosphate isomerase (GPI) were isolated from four representative E. histolytica strains belonging to zymodeme II, II!-, XIV, or XIX. Two alleleswere obtained from each strain; six alleles with eight polymorphic nucleotide positions were identified among the four strains. Two of these eightpolymorphic nucleotides resulted in non-conserved amino acid substitutions. Three GPI isoenzymes with distinct predicted isoelectric points wereidentified, which agrees well with the observed electrophoretic patterns of GPI from these strains. Amino acid comparisons of GPI from E. histolyticaand other organisms revealed that all amino acid residues implicated for substrate binding and catalysis were conserved. Biochemical characterizationof recombinant E. histolyticaGPI confirmed that it possessed kinetic parameters similar to GPI from other organisms. The electrophoretic mobility ofthree GPI isoenzymes was examined by starch gel electrophoresis. Thus, we have established the molecular basis of the classical isoenzymes patternsthat have been used for grouping E. histolytica isolates and for differentiation of E. histolytica from non-pathogenic Entamoeba dispar.© 2006 Elsevier Ireland Ltd. All rights reserved.

Keywords: Glucose phosphate isomerase; Entamoeba histolytica; Zymodemes

The protozoan parasite Entamoeba histolytica is an etiolog-ical agent of amoebiasis, causing an estimated 50 million casesof amebic colitis, dysentery, and extraintestinal abscesses [1] and40,000–100,000 deaths annually [2]. E. histolytica, togetherwith Giardia intestinalis, is categorized as a type I amito-chondriate protist, which lacks both typical mitochondria andhydrogenosomes [3] and is also deficient in all features ofaerobic metabolism. E. histolytica produces energy by glycol-ysis and fermentation under an anaerobic or microaerophilicenvironment [3,4]. In E. histolytica, all enzymes involved in the

conversion of glucose into pyruvate by glycolysis have beenidentified [5,6]. Molecular identification and enzymologicalcharacterization of 10 enzymes including hexokinase, inorganicpyrophosphate-dependent phosphofructokinase, and phospho-glucomutase were previously accomplished [7–11]. A recentstudy by Saarvedra et al. indicated that phosphoglyceratemutase, fructose-1,6-phosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate phosphate dikinaseare flux control steps because they possess the lowest catalyticefficiencies [11]. Four of about a dozen enzymes in theglycolytic pathway, namely hexokinase, phosphoglucomutase,glucose-6-phosphate isomerase (GPI), and malic enzyme, havebeen used to differentiate E. histolytica strains as well aspathogenic E. histolytica from non-pathogenic Entamoebadispar species. Although GPI, which is involved in the re-versible conversion of glucose-6-phosphate to fructose-6-phosphate, has been used as a gold standard to differentiatethe four major groups of E. histolytica strains (zymodemes II,II!-, XIV, and XIX) [12], the molecular basis of this

Parasitology International 55 (2006) 307–311www.elsevier.com/locate/parint

Abbreviations: GPI, glucose phosphate isomerase; EhGPI, Entamoebahistolytica GPI gene; EhGPI,Entamoeba histolytica glucose phosphate isomerase;ORF, open reading frame; PCR, polymerase chain reaction; rEhGPI, recombinantE. histolytica GPI.! Nucleotide sequence data reported in this paper are available in the EMBL,GenBank, and DDJB databases under the accession numbers BAE53444–BAE53451.! Corresponding author. Tel.: +81 27 220 8020; fax: +81 27 220 8025.E-mail address: nozaki@med.gunma-u.ac.jp (T. Nozaki).

1383-5769/$ - see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.parint.2006.08.001

Autho

r's p

erso

nal

copy

differentiation has not been established. In this study,we cloned andcharacterized GPI from representative isolates that belong to fourdistinct zymodemes (HM1:IMSS cl6, SAW1627, SAW755CRclB,and KU2 for zymodemes II, II!-, XIV, and XIX, respectively).

A putative EhGPI gene of the HM1:IMSS cl6 reference strainwas identified in the E. histolytica genome database (http://www.tigr.org/tdb/). It contains an open reading frame (ORF) of1641 nucleotides encoding a protein of 546 amino acids with apredicted molecular mass of 61.4 kDa and pI of 6.91. Toexamine polymorphisms of GPI sequences in E. histolyticastrains, the EhGPI ORF was amplified by PCR using genomicDNA from the four above-mentioned E. histolytica strains withsense (5"-CCTGGATCCGATGTTACCAACTCTTCCTGA-3")and antisense (5"-CCAGGATCCTTAGTTTTTTCTCATATCTTTAACA-3") primers designed based on the sequence infor-mation (engineered BamHI sites are underlined, and the start andstop codons are italicized). Conditions and parameters for PCRwere previously described [13]. PCR products were digestedwith BamHI and cloned into BamHI-digested pET-15b (Nova-gen) in the same orientation as the T7 promoter to produce a GPIfusion protein with a histidine tag at the amino terminus. Sixplasmid clones were randomly chosen for each E. histolyticastrain, and their inserts were sequenced on both strands.Nucleotide sequences of four GPI clones derived from HM1:IMSS cl6 were identical to the sequence in the database (XP650595 and AAT92031) and the sequence recently reported [11]and were designated “allele 1”. The remaining two clones con-

tained four nucleotide substitutions and were designated “allele2” (Fig. 1A). Similarly, two different alleles of GPI genes wereidentified from each of the three representative strains of otherzymodemes (II!-, XIV, and XIX) (Fig. 1A). These allelic genesequences (BAE53444–BAE53451) revealed heterogeneity ateight nucleotide positions. Altogether, six distinct alleles,designated “alleles 1–6” (Fig. 1A), were identified in the fourisolates. Two of these nucleotide polymorphisms resulted innon-conserved amino acid substitutions at positions 32 and 449,respectively, and distinct predicted isoelectric points. Isoelectricpoints of three allelic GPIs (designated isotypes 1–3) varied(isotype 1, 6.91; 2, 7.15; 3, 6.73) (Fig. 1A). However, thetotal number of amino acids and the predicted molecular massof all GPIs obtained from these four strains were identical.The predicted pIs of the two allelic gene products in thesestrains agree well with the observed mobility of GPI isoen-zymes in conventional starch gel electrophoresis, assuming thatE. histolytica GPI forms homo- and heterodimers as previouslyshown in other organisms (e.g., [14]).

To further examine features of E. histolytica GPIs (EhGPIs),they were compared with homologs from archaea, bacteria,fungi, protists, plants, and metazoa, revealing 30–64% iden-tity to EhGPI (representative species are shown in Fig. 1B).EhGPIs showed 44–60% identity to GPIs from Escherichiacoli, Aspergillus oryzae, Homo sapiens, and Spinacia oleracea.EhGPIs showed 41–54% identity to GPIs from other parasiticprotists including Leishmania mexicana, Trypanosoma brucei,

Fig. 1. Comparison of GPI nucleotide and protein sequences. (A) Schematic representation of nucleotide (top) and amino acid (bottom) sequences of two GPI alleles infour representative E. histolytica strains. Only nucleotides and amino acids showing heterogeneity are indicated, with predicted pI values. (B) Multiple alignments ofdeduced amino acid sequences of GPIs from E. histolytica and other organisms. Protein primary structures were aligned using the CLUSTALW program version 1.83(http://www.ddbj.nig.ac.jp/search/clustalw-e.html) with the BLOSUM matrix. Sequences are Entamoeba histolytica (Eh), Homo sapiens (Hs), Escherichia coli (Ec),Trypanosoma cruzi (Tc), T. brucei (Tb), Leishmania mexicana (Lm), Aspergillus oryzae (Ao), Plasmodium falciparum (Pf), Toxoplasma gondii (Tg), Spinaciaoleracea (So), Giardia intestinalis (Gi), and Trichomonas vaginalis (Tv). Asterisks indicate identical amino acids. Dots and colons indicate conserved amino acidssubstitutions. Dashes indicate computer-generated gaps. Residues deduced from crystal structures (16–18) to be important for substrate binding and/or catalysis ofGPIs are shaded in black, and the two signature patterns of the GPI family, [DENS]-X-[LIVM]-G-G-R-[FY]-S-[LIVMT]-X-[STA]-[PSAC]-[LIVMA]-G and [GS]-X-[LIVM]-[LIVMFYW]-XXXX-[FY]-[DN]-Q-X-G-V-E-X-X-K, are highlighted by gray boxes.

308 E. Razmjou et al. / Parasitology International 55 (2006) 307–311

Autho

r's p

erso

nal

copy

Trypanosoma cruzi, Toxoplasma gondii, and Plasmodiumfalciparum, while their similarity to representative amito-chondriate and microaerophilic/anaerobic protists Trichomo-

nas vaginalis and G. intestinalis was lower (31–32% identity).This finding suggests distinct GPI ancestors for theseamitochondriate parasites, which was also supported by the

Fig. 1 (continued ).

309E. Razmjou et al. / Parasitology International 55 (2006) 307–311

Autho

r's p

erso

nal

copy

previous phylogenetic study [15]. Two conserved signaturepatterns of GPI [16,17] are totally conserved in EhGPIs (Fig.1B). In addition, all conserved residues that were shown byvarious crystal structures or mutagenesis studies [18–20] to becrucial for substrate recognition, binding, or catalysis are highlyconserved in EhGPI (Fig. 1B). For instance, the followingamino acid residues are completely conserved: Thr205, Thr208,Ser153, Ser203, and Lys204 (numbering based on EhGPI),crucial for binding to the phosphate group; His382, Lys510,Glu210, Ile150, Thr378, Glu507, Arg266, Asp502, Gln503,and Glu351, essential for catalysis; and Gly151, Gly152,Gly264, and Gly265, involved in substrate specificity. EhGPIslack the long amino-terminal extension present in GPIs from L.mexicana, T. cruzi, and T. brucei that is involved in targetingthe enzyme to glycosomes [21]. This is consistent with thepremise that EhGPI is cytosolic. EhGPI also lacks an internalinsertion (between amino acids 227 and 228) present in GPIsfrom L. mexicana, T. cruzi, and T. brucei, a 27-amino-acidamino-terminal extension in G. intestinalis GPI, and a longcarboxyl-terminal extension present in GPI from T. vaginalis andG. intestinalis [22].

To investigate biochemical features of EhGPIs, the recom-binant EhGPIs corresponding to three representative proteins

[EhGPI isotypes 1–3, which represent allele 1 of HM1:IMSS cl6(BAE53444), allele 3 of SAW1627 (BAE53447), and allele 4 ofSAW755CR clB (BAE53448)] were produced using a prokary-otic expression system and purified as previously described [23].Upon SDS-polyacrylamide gel electrophoresis, the recombinantEhGPI (rEhGPI) proteins appeared as an apparently homoge-neous single band of approximately 63 kDa, which agrees withthe predicted size of a monomeric EhGPI protein with anadditional stretch of 21 amino acids at the amino terminus. Theformation of glucose-6-phosphate from fructose-6-phosphate inthe reverse reaction catalyzed by rEhGPI was measured bymonitoring the reduction of NADP+ spectrophotometrically at340 nm [24]. rEhGPI isotype 1 was active over a wide pH range(7.0–10.0) with optimal pH of 8.0–9.0 (Fig. 2A), consistent withthe previous study [11]. GPI activity was inhibited by addition ofa monovalent cation (e.g., Na+) (Fig. 2B), similar to GPI fromAspergillus niger [25]. rEhGPI followed Michaelis–Mentenkinetics. Kinetic constants under the standard assay conditions(containing 25–300 "M substrates; see Fig. 2 legend for details)were determined with Lineweaver-Burk plots. rEhGPI showed aKm value of 122±16 "M for fructose-6-phosphate and a specificactivity of 786±59 "mol min!1 mg protein!1 in the reversereaction, which are of the same order of magnitude as thosedetermined in the previous study (Vmax, 620 "mol min!1 mgprotein!1; Km, 480 "M; measured at pH 8 and 37 °C) [11]. TheKm and specific activity of rEhGPI were comparable to GPI fromother organisms, e.g., A. niger [25], L. mexicana [21,26], T.cruzi [27], T. brucei [26,28], and rabbit [28].

To establish the relationship between individual GPI isotypesidentified in this study and zymodemes revealed by starch gelelectrophoresis in four representative E. histolytica strains, weexamined both native and recombinant GPI isoenzymes usingstarch gel electrophoresis. The electrophoretic mobilities ofnative and recombinant GPI isoenzymes were similar despitemarginal differences in molecular mass and isoelectric point dueto the amino-terminal addition of the histidine tag (plus 2.4 kDa;6.91 vs. 6.88, 7.15 vs. 7.04, and 6.73 vs. 6.74 for EhGPI isotypes1–3, respectively) (data not shown), confirming that isoenzymeprofiles displayed by starch gel electrophoresis reflect two allelicGPI isotypes. Although zymodeme XIX showed the threeexpected bands on the starch gel, the mixture of recombinantGPI isotypes 1 and 3 gave rise to only two bands correspondingto two homodimers (isotypes 1/1 and 3/3) (data not shown). Thisis consistent with the hypothesis that a heterodimer (composedof isotypes 1 and 3) is not efficiently formed unless two GPIisotypes are simultaneously synthesized in vivo. These resultsmay also indicate that the homodimers are structurally stable.

These data demonstrate for the first time the molecular basisof classical isoenzyme patterns that have been used for thecharacterization of E. histolytica isolates by zymodeme analyses,considered the “gold standard” to differentiate E. histolyticastrains as well as pathogenic from non-pathogenic species[12,29]. Our results, together with the allelic heterogeneity ofhexokinase and phosphoglucomutase [7–10], indicate that iso-enzyme profiles are primarily, if not solely, attributable to se-quence diversities at the primary sequence level. Our data furtherenable the establishment of PCR-based zymodeme analysis that

Fig. 2. (A) pH dependence of E. histolytica GPI. The pH optimum of theenzyme was determined in the direction of glucose-6-phosphate formation fromfructose-6-phosphate between pH 5.5 and 11 at 25 °C in a coupling assay. The100-"l assay mixture was comprised of 100 mM either 2-Morpholinoethane-sulfonic acid (pH 5.5, 6.0, and 6.5), N-(2-hydroxyethyl)piperazine-N"-(2-ethanesulfonic acid) (pH 7.0, 7.5, and 8.0), N-[Tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid (pH 8.5, 9.0, and 9.5), or 3-(cyclohexylamino)-1-propanesulfonic acid (pH 10, 10.5, and 11); 2 mM fructose-6-phosphate;0.5 mM NADP; 0.1 U glucose-6-phosphate dehydrogenase; and 0.1 "g purifiedEhGPI protein. Error bars represent standard errors from three independentexperiments. (B) The effect of NaCl on EhGPI activity. The activity of EhGPIwas determined in a 100-"l assay mixture comprised of 100 mM TAPS, pH 9.0;2 mM fructose-6-phosphate; 0.5 mM NADP; 0.1 U glucose-6-phosphatedehydrogenase; and 0.1 "g purified EhGPI protein, with the addition of 0–500 mM NaCl. Error bars represent standard errors from five independentexperiments.

310 E. Razmjou et al. / Parasitology International 55 (2006) 307–311

Autho

r's p

erso

nal

copy

can replace laborious and time-consuming zymodeme analysisusing starch gel electrophoresis.

Acknowledgments

The authors express their appreciation to Vahab Ali, GunmaUniversity; Yasuo Shigeta and Yumiko Saito-Nakano, NationalInstitute of Infectious Diseases, Japan; Bahram Kazemi,Shaheed Beheshti University of Medical Sciences; and BijanFarzami, Tehran University of Medical Sciences for technicalassistance and helpful discussions. This work was supported inpart by a Grant-in-Aid for Scientific Research from the Ministryof Education, Culture, Sports, Science, and Technology of Japanto T.N.; a grant for Research on Emerging and Re-emergingInfectious Diseases from the Ministry of Health, Labor, andWelfare; a grant for the Project to Promote the Development ofAnti-AIDS Pharmaceuticals from the Japan Health SciencesFoundation to T.N.; a fellowship from Japan Society for thePromotion of Science to A.H.; a scholarship from The Ministryof Health, Treatment, andMedical Education of Iran to E.R.; andfinancial support from Tehran University to M.R.

References

[1] Gilchrist CA, Petri WA. Virulence factors of Entamoeba histolytica. CurrOpin Microbiol 1999;2:433–7.

[2] WHO/UNESCO Report. A consultation with experts on amoebiasis.Epidemiol Bull 1997;18:13–4.

[3] Müller M. Enzymes and compartmentation of core energy metabolism ofanaerobic protists—a special case in eukaryotic evolution. In: CoombsGH, Vickerman K, Sleigh MA, Warren A, editors. Evolutionary Rela-tionships among Protozoa. The Netherlands: Kluwer Academic Publish-ers; 1998. p. 109–32.

[4] Reeves RE. Metabolism of Entamoeba histolytica Schaudinn. AdvParasitol 1903;23:105–42.

[5] Loftus B, Anderson I, Davies R, Alsmark UC, Samuelson J, Amedeo P, et al.The genome of the protist parasite Entamoeba histolytica. Nature2005;433:865–8.

[6] Anderson IJ, Loftus BJ. Entamoeba histolytica: observations on metabo-lism based on the genome sequence. Exp Parasitol 2005;110:173–7.

[7] Ortner S, Plaimauer B, Binder M, Scheiner O, Wiedermann G, DucheneM. Molecular analysis of two hexokinase isoenzymes from Entamoebahistolytica. Mol Biochem Parasitol 1995;73:189–98.

[8] Ortner S, Clark CG, Binder M, Scheiner O, Wiedermann G, Duchene M.Molecular biology of the hexokinase isoenzyme pattern that distinguishespathogenic Entamoeba histolytica from nonpathogenic Entamoeba dispar.Mol Biochem Parasitol 1997;86:85–94.

[9] Ortner S, BinderM, Scheiner O,WiedermannG, DucheneM.Molecular andbiochemical characterization of phosphoglucomutases from Entamoebahistolytica and Entamoeba dispar. Mol Biochem Parasitol 1997;90:121–9.

[10] Kroschewski H, Ortner S, Steipe B, Scheiner O, Wiedermann G, DucheneM. Differences in substrate specificity and kinetic properties of therecombinant hexokinases HXK1 and HXK2 from Entamoeba histolytica.Mol Biochem Parasitol 2000;105:71–80.

[11] Saavedra E, Encalada R, Pineda E, Jasso-Chavez R, Moreno-Sanchez R.Glycolysis in Entamoeba histolytica. Biochemical characterization ofrecombinant glycolytic enzymes and flux control analysis. FEBS J2005;272:1767–83.

[12] Sargeaunt PG, Williams JE, Grene JD. The differentiation of invasive andnon-invasive Entamoeba histolytica by isoenzyme electrophoresis. TransR Soc Trop Med Hyg 1978;72:519–21.

[13] Nozaki T, Asai T, Sanchez LB, Kobayashi S, Nakazawa M, Takeuchi T.Characterization of the gene encoding serine acetyltransferase, a regulatedenzyme of cysteine biosynthesis from the protist parasiteEntamoeba histolyticaand Entamoeba dispar: regulation and possible function of the cysteinebiosynthetic pathway in Entamoeba. J Biol Chem 1999;274:32445–52.

[14] Van Beneden RJ, Powers DA. Structural and functional differentiation oftwo clinally distributed glucose phosphate isomerase allelic isozymes fromthe teleost Fundulus heteroclitus. Mol Biol Evol 1989;6:155–70.

[15] Huang J, Mullapudi N, Lancto CA, Scott M, Abrahamsen MS, KissingerJC. Phylogenomic evidence supports past endosymbiosis, intracellular andhorizontal gene transfer in Cryptosporidium parvum. Genome Biol2004;5:R88.

[16] Sun YJ, Chou CC, Chen WS, Wu RT, Meng M, Hsiao CD. The crystalstructure of a multifunctional protein: phosphoglucose isomerase/autocrinemotility factor/neuroleukin. Proc Natl Acad Sci U S A 1999;96:5412–7.

[17] Hansen T, Oehlmann M, Schonheit P. Novel type of glucose-6-phosphateisomerase in the hyperthermophilic archaeonPyrococcus furiosus. J Bacteriol2001;183:3428–35.

[18] Lee JH, Chang KZ, Patel V, Jeffery CJ. Crystal structure of rabbit phospho-glucose isomerase complexed with its substrate D-fructose 6-phosphate.Biochemistry 2001;40:7799–805.

[19] Jeffery CJ, Hardre R, Salmon L. Crystal structure of rabbit phosphoglucoseisomerase complexed with 5-phospho-D-arabinonate identifies the role ofGlu357 in catalysis. Biochemistry 2001;40:1560–6.

[20] Cordeiro AT, Godoi PH, Silva CH, Garratt RC, Oliva G, Thiemann OH.Crystal structure of human phosphoglucose isomerase and analysis of theinitial catalytic steps. Biochim Biophys Acta 2003;1645:117–22.

[21] Cordeiro AT, Michels PA, Delboni LF, Thiemann OH. The crystal structureof glucose-6-phosphate isomerase from Leishmania mexicana revealsnovel active site features. Eur J Biochem 2004;271:2765–72.

[22] Henze K, Horner DS, Suguri S, Moore DV, Sanchez LB, Muller M, et al.Unique phylogenetic relationships of glucokinase and glucose phosphateisomerase of the amitochondriate eukaryotes Giardia intestinalis, Spiro-nucleus barkhanus and Trichomonas vaginalis. Gene 2001;281:123–31.

[23] Ali V, Shigeta Y, Tokumoto U, Takahashi Y, Nozaki T. An intestinalparasitic protist Entamoeba histolytica possesses a non-redundant NIF-likesystem for iron–sulfur cluster assembly under anaerobic conditions. J BiolChem 2004;279:16863–74.

[24] Jeong JJ, Fushinobu S, Ito S, JeonBS, ShounH,Wakagi T. Characterizationof the cupin-type phosphoglucose isomerase from the hyperthermophilicarchaeon Thermococcus litoralis. FEBS Lett 2003;535:200–4.

[25] Ruijter GJ, Visser J. Characterization of Aspergillus niger phosphoglucoseisomerase. Use for quantitative determination of erythrose 4-phosphate.Biochimie 1999;81:267–72.

[26] Nyame K, Do-Thi CD, Opperdoes FR, Michels PA. Subcellulardistribution and characterization of glucose phosphate isomerase inLeishmania mexicana mexicana. Mol Biochem Parasitol 1994;67:269–79.

[27] Concepcion JL, Chataing B, Dubourdieu M. Purification and properties ofphosphoglucose isomerases of Trypanosoma cruzi. CompBiochemPhysiolB Biochem Mol Biol 1999;122:211–22.

[28] Marchand M, Kooystra U, Wierenga RK, Lambeir AM, Van Beeumen J,Opperdoes FR, et al. Glucose phosphate isomerase from Trypanosomabrucei. Cloning and characterization of the gene and analysis of the enzyme.Eur J Biochem 1989;184:455–64.

[29] Sargeaunt PG. Zymodemes of Entamoeba histolytica. In: Ravdin JI, editor.Amoebiasis: human infection by Entamoeba histolytica. New York: JohnWiley and Jone Inc; 1988. p. 370–87.

311E. Razmjou et al. / Parasitology International 55 (2006) 307–311