- Email: [email protected]
Application of single strand conformational polymorphism (SSCP) analysis with fluorescent primers for differentiation of Schistosoma haematobium group species
Diagnosis Application fluorescent species R. A. Kane’, Wellcome London, Merelbeke,
of single strand conformational polymorphism (SSCP) analysis with primers for differentiation of Schistosoma haematobium group
J. Bartley’,
J. R. Stothard’,
J. Vercruysse
Biomedical Laboratories, Biomedical Parasitology UK; ‘Faculteit Diergeneeskunde, Departement Belgi2
‘, D. Rollinson’
Division, Virologie,
Department
Parasitologic
lWolfson
and V. R. Southgate’
of Zoology, The Natural History en Immunologic, Universiteit
Museum, van Gent,
Abstract To assess the utility of single-stranded conformational polymorphism (SSCP) analysis for the differentiation of schistosomes. using methods adauted for a Perkin Elmer ABI Prism 377TM automated seauencer. 3 isolates of &l&o&ma hiematobium, 2*of S. intercalatum and single isolates of S. curassoni and-S. bov& were selected for study. Two fluorescently labelled, double-stranded polymerase chain reaction products, amplified from the mitochondrial cytochrome oxidase subunit 1 (COl) gene and the nuclear ribosomal second internal transcribed spacer (ITS2), were generated from single male and female worms. Changes in electrophoretic mobility of fragments within an SSCP profile revealed variation at individual, isolate and species levels. The mutational basis between representative SSCP profiles was confirmed by direct sequencing, demonstrating that single point substitutions were detectable. SSCP analysis has considerable potential as an alternative molecular method of identification and characterization of schistosomes. More broadly, fluorescence-based SSCP analysis is applicable to almost any gene target from any species of parasite and is a powerful molecular tool for genetic profiling. Keywords: schistosomiasis, Schistosoma bovis, Schistosoma curassoni, Schistosoma haematobium, turn, identification, single-stranded conformational polymorphism analysis, genetic profile
Introduction Schistosomiasis is a widespread parasitic disease in tropical and sub-tropical regions of the world (SAVIOLI et al., 1997). Of the 19 recognized species of schistosomes, 5 are important parasites of humans and are placed within 3 of the 4 species groups of Schistosoma: S. haematobium group (S. haematobium and S. intercalaturn), S. manson; group (S. mansonz) and S. japonicum group (S. japonicum and S. mekongi). The groups are largely defined by phenotypic characters such as adult worm and egg morphology and intermediate host specificity (ROLLINSON&SOUTHGATE,~~~~). Throughout much of Africa, both urinary and intestinal forms of schistosomiasis occur (ROLLINSON & JOHNSTON, 1996). Urinary schistosomi&is is attributable to infection with S. haematobium whilst infection with either S. mansoni or S. intercalatum results in intestinal schistosomiasis. S. mansoni is more widespread throughout Africa than 5’. intercalatum, which has restricted distribution limited to the Democratic Republic of Congo (formerly Zaire), Equatorial Guinea, Gabon, Nigeria and Cameroon with isolated case reports from other parts of central and western Africa (BROWN et al., 1984). S. intercalatum is also found in SZo Torn& (SOUTHGATE et al., 1994). Two distinct strains of S. intercalatum are recognized, having different phenotypic characteristics and geographical distribution. S. haematobium has a wide distribution in Africa; its compatibility with species of Bulinus, the snail intermediate host, is varied and often limited to specific interactions confined to a given locality (ROLL INSON STON,
&
SOUTHGATE,
1987;
ROLLINSON
& JOHN-
1996). Other species within the S. haematobium group are of veterinary importance, including S. bovis, S. curassoni and S. mattheei; in certain areas of Africa (e.g., Sudan) S. bovis causes serious problems in livestock (ROLLINSON~LSOUTHGATE, 1987).
Address for correspondence: Mr R. A. Kane, Wolfson Wellcome Biomedical Laboratories, The Natural History Museum, Cromwell Road, London, SW7 5BD, UK; phone +44 (0)207 942 5152; fax +44 (0)207 942 5347, e-mail [email protected]
Schistosoma
intercala-
In marked contrast to the majority of Digenea, the schistosomes are dioecious and have successfully colonized the blood vasculature of vertebrates. If they are able to infect the same definitive host, there appear to be few, if any, physiological barriers preventing encounter or mating of schistosome worms of different species (SOUTHGATE et al., 1998). Hence, there are cases of hybridization in nature, especially where transmission cycles are sympatric, favouring the occurrence of mixed infections in the mammalian host. Within the S. haematobium group, several species are known to have the potential to hybridize but, interestingly, viable crosses are not always reciprocal (SOUTHGATE & ROLLINSON, 1987). Whilst hybridization between schistosome species belonging to different species groups does not occur, interspecific interactions do. For example, mixed infections of S. haematobium and S. mansoni in laboratory animals readily form heterospecific and homospecific worm pairs. S. haematobium males exhibit a greater ability to take S. mansoni females away from S. mansoni males, perhaps an indication of their greater competitiveness (WEBSTER et al., 1999). In such heterospecific pairings, identification of female worms is relatively straightforward by the examination of intrauterine egg morphology or the position of the ovary. Identification of male worms, however, is more problematic and is not easily determined by morphology. The development of better molecular methods for identification and characterization of schistosome populations will alleviate logistical problems associated with current methods and provide data useful for population genetics and epidemiology. Natural hybrids can be detected and monitored by possession of intersecting sets of molecular characters. In addition, by comparison of mitochondrial deoxyribonucleic acid (mtDNA) and nuclear DNA loci, the maternal/paternal genealogy of the hybridizing species can be determined, assuming strict maternal inheritance of mtDNA and Mendelian assortment at nuclear loci. Since the development of the single-stranded conformational polymorphism (SSCP) method by ORITA et al. (1989), the technique has become widespread within the field of medical research for mutational analysis. GASSER &
R. A. KANE
S11236
ZHU (1999) have reviewed SSCP and other mutation detection techniques based on the polymerase chain reaction (PCR) as applied to parasites, concluding that the methods have advantages over more conventional DNA methods, provided they are selected with care. Previous SSCP studies have relied upon radioactive labelling or silver staining methods (HISS et al., 1994; DE LEON et al., 1998; G~SER et al., 1998; STOTHARD et al.. 1997. 1998: B~GH et al.. 1999; LIU et al., 1999). In this pap& the ðodology;s carried a stage-further, taking advantage of modern fluorescent chemistry using an ABI Prism 377TM automated sequencer. Four Schistosoma species, including 3 isolates of S. haematobium and 2 isolates of S. intercalatum, were examined to determine whether SSCP analysis can generate informative markers. Two genetic loci from the mitochondrion and nucleus were targeted. A 177 bp fragment was studied from the mitochondrial cytochrome oxidase subunit 1 (COl) gene, and a 207 bp fragment from the nuclear ribosomal spacer ITS2. Methods Generation offluorescent PCR fragments Six individual worms from each of the 4 snecies and 7 isolates were selected for study (see the Table). DNA was extracted according to the method outlined by WALKER et al. (1986) with minor modification. PCR products for CO1 were generated using Amplitaq GoldTM nolvmerase (Perkin Elmer) and the following cycling don&ions oh a HybaidTti thermal cycler: i cycle of 10 min at 94”C, 1 min at 5O”C, and 1 min at 72”C, 30 cycles of 1 min at 94”C, 1 min at 5O”C, and 1 min at 72”C, and a final cycle of 1 min at 94”C, 1 min at 50°C and 10 min at 72°C. The sequence of the forward primer, COlSSCPl, was as follows: 5’GTT CWGTGACTATGATTAT3’ and the reverse mimer. cOlSSCP2,5’CTAATGAAGAAGCRG&GC3 The forward primer was fluorescently labelled at the 5’ end with NED (yellow) and the reverse primer with HEX (green). The ITS2 fragment was amplified using a Perkin ElmerTM 9700 cycler and Amplitaq GoldTM. The cycling programme consisted of initial denaturation for 10 min at 94”C, followed by 30 cycles of 1 min at 94”C, 1 min at 58”C, and 1 min at 72°C. There was a final elongation period of 10 min at 72°C. The forward primer, ITSSSCPl, had the sequence 5’GCA TATCAACGCGGGS’ and was labelled at the 5’ end with NED, while the sequence of the reverse primer, ITSSSCP2, was 5’ACAAACCGTAGACCGAACC3’. The latter was labelled at the 5’ end with HEX. All PCR nroducts were nurified using Nucleon OCTM clean-;p kits (Amershim Life Sciencu). Electrophoretic conditions and sample preparation SSCP analysis on an ABI Prism 377TM automated sequencer was performed using a Perkin Elmer Applied Biosystems protocol, but with the following specific modifications and run conditions. Electrophoretic gel. Non-denaturing 5% Long RangerTM gel prepared as follows: 1.25 g glycerol, 8.1 mL Table.
Origins
of isolates
Species
Country
S. S. S. S. S. S. S.
Senegal Mali Mauritius SBo Tom& Cameroon Senegal Senegal
haematobium haematobium haematobium intercalatum intercalatum curassoni bovis
used in this study NHM isolate number 3572 3125 3135 2758 1970 2517 B2
Males Females G 3 3 3 6
3 3 3 3 6 -
ETAL.
50% Long Ranger gel solution concentrate (FMC), and 30 mL distilled water. The volume was adjusted to 45 mL with distilled water, and the solution was filtered and added to 5 mL of filtered 10X tris-borate-ethylenediaminetetraacetic acid (TBE) buffer at pH 8.3. The whole was then degassed for 5 min and 250 & of 10% ammonium persulphate and 35 & of N,N,N’,N’-tetramethylethylenediamine (TEMED) were added. The resultant solution was mixed and poured into the assembled gel plates. Pre-run module on 377 DNA Sequencer. Electrophoresis voltage 1000 V. current 35 mA. Dower 50 W, nel tempera&e 20°C (iemperatures were-maintained -wyt.h an external Multitemp IIITM thermostatic circulator [Pharmacia Biotech]). The gel was pre-run for 5 min. Run module on 377 DNA Sequencer. Electrophoresis voltage 2140 V, current 60 mA, power 200 W, gel temperature 20°C. The data collection time was 13 h. Sample preparation. A 60X mix consisting of 120 ti of dei&&d~formamide containing Blue Dextran and 2.1 uL of 1 M NaOH. 2 UL of this mix were added to 1 & of appropriately d&ted PCR product and 0.75 pL of GS2500 internal lane standard (Perkin Elmer), giving a total sample volume of 3.75 &. These samples were then heated at 95°C for 5 min and placed immediately on ice. 2 & of each sample were loaded and run into the gel for 3 min using the pre-run settings before this was terminated and the full run module settings implemented. Internal lane standards were run within each sample and, because the standards were denatured together with the experimental DNA, measurement of mobility in terms of exact nucleotide length was not possible. Mobility values were therefore derived by the use of scan numbers, which are recorded by the sequencer throughout the run. All DNA sequences obtained in this study were ‘blasted’ (ALTSCHUL et al., 1990) against the EMBL database in order to ensure that they were of schistosomal origin. Results Electronic images of typical SSCP profiles for CO1 from individual worms are shown in Fig. 1A. The coloured banding patterns correspond to single stranded DNA fragments that possess either green or yellow fluorescent primers. It is immediately apparent that the reverse green strand is travelling in advance of the complementary yellow forward strand for all the species and isolates used in the study. The SSCP profile could not differentiate S. haematobium isolates from Senegal and Mali, and subsequently their DNA sequences were shown to be identical. There was, however, a notable difference between the profile obtained with S. haematobium from Mauritius and those from Senegal and Mali. Sequencing the CO1 fragment revealed that the difference in mobility was presumably att’ributed to a single substitution of thymidine (T) for cytidine (C) located 38 bp from its 3’ end and 16 bp upstream of the COlSSCP2 priming site (see Fig. 1B). This was a synonymous substitution, the amino acid, glycine, remaining unaltered. The chroinatographic profiles of the faster migrating green strand in all cases indicated a doublet, whilst the slower migrating yellow strand was shown to be a single peak. The only exceptions were the Mauritian isolate- (a triplet), and thk 4 slower mizrants of S. bovis (doublets) (see Fia. 2). In the la&, the two faster moving s&an&, in &a&s 38 and 41, differed in sequence from their neighbours by the substitution of a C for a T, 7 bp upstream from the primer COlSSCP2 and only 9 bp downstream from the similar substitution in the Mauritian S. haematobium isolate. Again, another synonymous mutation was detected, the amino acid threonine remaining un-
SSCP
ANALYSIS
OF SCHISTOSOMA
SW.
9300
9500
2517
9600
37##
CO1 chromatograms
9400 9800
4900
1QQQQ
1
ITS2 chromatograms
10200
1#4##
10600
10800
Fig. 2. Selection of chromatograms showing peak structures for the various species of Schistosoma and variable isolates. Where one isolate within a species differs in format from another, both forms are shown. In the case of S. bovis, the 2 variant forms within isolate B2 are overlapped. The aberrant S. intercalatum sample is identified by the superscript number 27 next to the NHM coding. Yellow peaks are shown in black for visual clarity. The isolate code numbers are explained in the Table. Consult the scan reading at the top of each chromatographic ‘snapshot’ to compare mobility values as the frames are not all at the same scale.
10 9200
####
$
3 q
SSCP
ANALYSIS
OF SCHISTOSOMA
SW.
S11239
II
*
.
-
4 ii
. . *
.
. . .
s11240
changed. Sample number 27, an S. intercalatum isolate from Cameroon, had an extra fragment in both green and yellow strands (see Fig. 1A). The chromatogram revealed a ‘marginal’ triplet for the reverse strand and a doublet for the forward strand (see Fig. 2). When sequenced, however, the CO1 fragment from this worm proved to be the same as that of the S. intercalaturn isolate from S5o Tome. It is therefore not certain whether the additional peaks observed were genuinely different conformations or, perhaps, an artefact. The area of a chromatographic peak may be used to determine the relative quantity of single stranded DNA present in a particular band. This area can vary slightly from track to track depending on the original concentration of DNA loaded; however, the overall proportion should remain relatively the same. Thus, using the reverse strand for the 6 individual worms of S. bovis as an example, a relative value for the percentage of single stranded molecules adopting a particular conformation can be obtained. In this case an average value for the 6 worms was calculated, revealing that 20% of the molecules adopted the structure associated with the smaller fast-moving peak, while 80% arranged themselves in a manner corresponding to the larger peak. The electronic image of the ITS2 fragment generated from the same worms revealed fewer variants and showed a completely different profile to that of CO 1, as might be expected. In this case, the forward and reverse strands had changed positions according to the species studied. All the S. haematobium PCR fragments had faster migrating yellow forward strands, while in the remaining species this strand migrated more slowly than the green counterpart (see Fig. 3A). Sequencing showed that the difference in mobility between S. haematobium and the other 3 species was due to substitutions at 3 sites. These were located as follows from the 5’ end of the fragment: position 25, where in S. haematobium adenosine (A) is replaced bv_ -guanosine (G); position 80, where T changes to C; and position 130. where A is anain substituted bv G (see Fig. 3B). In all &her positions, the sequences here‘ the sime. ‘The location of nucleotide variants towards the end regions of either the CO1 or ITS2 fragments did not appear to inhibit their detection by this method. Discussion
SSCP analysis has been based on the premise that single-stranded DNA molecules adopt unique secondary structural conformations when electrophoresed under non-denaturing conditions (ORITA et al., 1989; HISS et al., 1994; STOTHARJIet al., 1997; GASSER& ZHU, 1999). The exact nature of a particular conformation is determined by the single strand length, nucleotide sequence, and location of intra-strand pairing. Recent evidence has also shown that tertiary structures (i.e., sugar-base and sugar-sugar interactibns) are an additional determinant of mobility (LIU et al., 1999). The sensitivity of the technique is impressive, for example single point mutations w&-e detectable &thin CO1 between isolates of S. haematobium. Similarly for ITS2, where the striking difference between isolates of S. haematobium and the remaining schistosome species was attributable to just 3 point mutations. Sensitive molecular methods are needed to monitor tb.e genetic epidemiology of schistosome populations (BO*LES et al., 1993,-i995; DEW&S et-al, 1992). New techniaues for identification of moruholoeicallv similar cercaiiae would be particularly val
R. A. KANE ETAL.
intercalatum, S. cursassoni and S. bovis, and the clear differentiation of these species from S. haematobium, support previous phylogenetic work that has shown a closer evolutionary affinity among S. intercalatum, S. curassoni and S. bobis thanbetweenthese species and S. (see KANE & ROLLINSON,1994; ROLLINSON et al., 19971. Molecular methods that enable haematobium
identification of hybrid strains are particularly important. Introgression of genes from one species into another could potentially facilitate transfer of resistance factors from control-selected populations into naive populations or vice versa, or even alter intermediate host compatibility if this trait was under genetic control. Whilst previous studies employing SSCP analysis have used conventional electrophoretic apparatus and methods of detection, greater resolution and more information are obtainable through the utilization of an ABI Prism 377TM automated sequencer. As each PCR primer can be labelled with a separate fluorescent dye, both positive and negative strands can be simuitaneouslv identified and analvsed with GenescanTM and Geno&erTM software. Labilling the two DNA strands with different colours ensures that the same strands are compared across samples, thereby removing false comuarisons. The SSCP nrofiles of ITS2 are a g.ood example, where mobility 0; positive and negativeitrands can be highly divergent; without the use of 2 colours, the banding patterns could be confused. The display of both fluorescent colours together also indicates that a fragment is comprised of both strands. In addition, colour labelling permits multiplexing of different PCR products into a single lane, which can reduce both processing time and cost. Manipulation and storage of mobility data are greatly simplified with GenescanTM and GenotyperTM software. The chromatograms generated allow greater resolution between samples, minimizing the potential errors of visual interpretation, and the use of internal lane mobility standards enables adjustment between lanes. Acknowled!xements
We thank-Mr Stephen L. Hughes of the Veterinary Laboratory Agency for his assistance with GenotvpeP. We congratulate Dbuglas Barker on his long career-and contribution to trooical medicine. and wish him everv hauoiness during his retirement. Thanks* are due to the &rop,an Comm&ity (award ICI S-C196-0041) and the Wellcome Trust for finahcially supporting the work. References
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular
Biology,
215, 403-410.
BQgh, H. O., Zhu, X. Q., Qian, B. Z. & Gasser, R. B. (1999). Scanning for nucleotide vanations in mitochondrial DNA fragments of Schistosoma japonicum by single-strand conformation
polymorphism.
Parasitology,
118, 73-82.
Bowles, J., Hope, M., Tiu, W. U., Liu, X. S. & McManus, D. P. (1993). Nuclear and mitochondrial genetic markers highly conserved between Chinese and Philippine Schistosoma japonicum. Acta Tropica, 55, 217-229. Bowles, J., Blair, D. & McManus, D. P. (1995). A molecular phylogeny of the human schistosomes. Molecular Phylagenetics and Evolution, 4, 103-109. Brown, D. S., Sarfati, C., Southgate, V. R., Ross, G. C. & Knowles, R. J. (1984). Observations on Schistosoma intercalatum in south-east Gabon. Zeitschrift fiir Parasitenkunde, 70, 243-253. De Leon, M. I’., Yanagi, T., Kikuchi, M., Mu, J., Ayau, O., Matta, V.. Paz. M.. Tuarez. S.. Kanbara. H.. Tada. I. & Hirayama; K. (i 998). “Chara&e&ation of ?ryp&oso& cruzi populations by DNA polymorphism of the cruzipain gene detected by single-stranded DNA conformation polymorphism (SSCP) and direct sequencing. International ‘foumal for Pa,&olo& 28, 1867-187a. Desn&s. L., Imbert-Establet, D., Combes, C. & Bonhomme, F: (1492). Molecular evidence-linking hominid evolution td
SSCP
ANALYSIS
OF SCHISTOSOMA
SW.
recent radiation of schistosomes (Platyhelminthes: Trematoda). Molecular Phylogenetics and Evolution, 1,295-304. Gasser, R. B. & Zhu, X. Q. (1999). Sequence-based analysis of enzymatically amplified DNA fragments by mutation detection techniques. Parasitology Today, 15,462-465. Gasser, R. B., Zhu, X. Q. & McManus, D. P. (1998). Display of sequence variation in PCR-amplified mitochondrial DNA regions of Echinococcus by single-strand conformation polymorphism. Acta Tropica, 71, 107-l 15. Hiss, R. H., Norris, D. E., Dietrich, C. H., Whitcomb, R. F., West, D. F., Bosio, C. F., Kambhampati, S., Piesman, J., Antolin, M. F. & Black, W. C. I. V. (1994). Molecular taxonomy using single-strand conformation polymorphism (SSCP) analysis of mitochondrial ribosomal DNA genes. Insect Molecular Biology, 3, 17 I- 182. Kane, R. A. & Rollinson, D. (1994). Repetitive sequences in the ribosomal DNA internal transcribed spacer of Schistosoma haematobium, Schistosoma intercalatum and Schistosoma Molecular and Biochemical Parasitology, 63, mattheei. 153-156. Liu, Q., Feng, J., Buzin, C., Wen, C., Nozari, G., Mengos, A., Nguyen, V., Liu, J., Crawford, L., Fujimura, F. K. & Sommer, S. S. (1999). Detection of virtually all mutations-SSCP (DOVAM-S): a rapid method for mutation scanning with virtually 100% sensitivity. BioTechniques, 36, 58-63. Orita, M., Iwahana, H., Kanazawa, H., Hayashi, K. & Sekiya, T. (1989). Detection of polymorphisms of human DNA bv gel electrophoresis as single-strand conformation pol& morphisms. Proceedings of the National Academy of Sciences of the USA, 86,2766-2770. Rollinson, D. & Johnston, D. A. (1996). Schistosomiasis: a persistent parasitic disease. Interdisciplinary Science Reviews, 21,140-154. Rollinson, D. & Southgate, V. R. (1987). The genus Schistosoma: a taxonomic appraisal. In: The Biology of Schistosomes: from genes to latrines. Rollinson, D. & Simpson, A. J. G. (editors). London &New York: Academic Press, pp. l-49. Rollinson, D., Kaukas, A., Johnston, D. A., Simpson, A. J. G. & Tanaka, M. (1997). Some molecular insights into schisto-
S11241
some evolution. International Journal for Parasitology, 27, 11-28. Savioli, L., Renganathan, E., Montresor, A., Davis, A. & Behbehani, K. (1997). Control of schistosomiasis-a global picture. Parasitology Today, 13,444-448. Southgate, V. R. & Rollinson, D. (1987). Natural history of transmission and schistosome interactions. In: The Biology of Schistosomes:fiom genes to latrines. Rollinson, D. & Simpson, A. 1. G. (editors). London & New York: Academic Press. pp:347-378. Southgate, V. R., Rollinson, D., Kaukas, A., Almeda, J., Sousa, A. M., Castro, F., Soares, E. & Coarachan, M. (1994). Schistosomiasis in the Republic of Slo Tome and Princiue: characterization of Schistosoma intercalatum. Transactions of the Royal Socie y of Tropical Medicine and Hygiene, 88,479-486. Southgate, V. R., Jourdane, J. & Tchuem Tchuentt, L. A. (1998). Recent studies on the reproductive biolow of the schistosomes and their relevance to speciation in the Digenea. InternationalJoumalfor Parasitology, 28, 1159-1172. Stothard, J. R., Frame, I. A. & Miles, M. A. (1997). Use of polymerase chain reaction-based single strand conformational polymorphism and denaturing gradient gel electrophoresis methods for detection of sequence variation of ribosomal DNA of Typanosoma cruzi. International Journal for Parasitology, 27, 339-343. Stothard, J. R., Yamamoto, Y., Cherchi, A., Garcia, A. L., Valente. S. A. S.. Schofield C. 1. & Miles. M. A. 11998’1. A preliminary survey of mitochondrial sequence variation &I Triatominae (Hemiptera: Reduviidae) using polymerase chain reaction reaction-based single strand conformational polymorphism (SSCP) analysis and direct sequencing. Bulletin of Entomological Research, 88, 553-560. Walker, T. K., Rollinson, D. & Simpson, A. J. G. (1986). Differentiation of Schistosoma haematobium from related species using cloned ribosomal RNA gene probes. Molecular and Biochemical Parasitology, 20, 123- 13 1. Webster, B. L., Southgate, V. R. & Tchuem Tchuente, L. A. (1999). Mating interactions between Schistosoma haematobium and S. mansoni. Journal of Helminthology, 73, 351-356.
of single strand conformational polymorphism (SSCP) analysis with primers for differentiation of Schistosoma haematobium group
J. Bartley’,
J. R. Stothard’,
J. Vercruysse
Biomedical Laboratories, Biomedical Parasitology UK; ‘Faculteit Diergeneeskunde, Departement Belgi2
‘, D. Rollinson’
Division, Virologie,
Department
Parasitologic
lWolfson
and V. R. Southgate’
of Zoology, The Natural History en Immunologic, Universiteit
Museum, van Gent,
Abstract To assess the utility of single-stranded conformational polymorphism (SSCP) analysis for the differentiation of schistosomes. using methods adauted for a Perkin Elmer ABI Prism 377TM automated seauencer. 3 isolates of &l&o&ma hiematobium, 2*of S. intercalatum and single isolates of S. curassoni and-S. bov& were selected for study. Two fluorescently labelled, double-stranded polymerase chain reaction products, amplified from the mitochondrial cytochrome oxidase subunit 1 (COl) gene and the nuclear ribosomal second internal transcribed spacer (ITS2), were generated from single male and female worms. Changes in electrophoretic mobility of fragments within an SSCP profile revealed variation at individual, isolate and species levels. The mutational basis between representative SSCP profiles was confirmed by direct sequencing, demonstrating that single point substitutions were detectable. SSCP analysis has considerable potential as an alternative molecular method of identification and characterization of schistosomes. More broadly, fluorescence-based SSCP analysis is applicable to almost any gene target from any species of parasite and is a powerful molecular tool for genetic profiling. Keywords: schistosomiasis, Schistosoma bovis, Schistosoma curassoni, Schistosoma haematobium, turn, identification, single-stranded conformational polymorphism analysis, genetic profile
Introduction Schistosomiasis is a widespread parasitic disease in tropical and sub-tropical regions of the world (SAVIOLI et al., 1997). Of the 19 recognized species of schistosomes, 5 are important parasites of humans and are placed within 3 of the 4 species groups of Schistosoma: S. haematobium group (S. haematobium and S. intercalaturn), S. manson; group (S. mansonz) and S. japonicum group (S. japonicum and S. mekongi). The groups are largely defined by phenotypic characters such as adult worm and egg morphology and intermediate host specificity (ROLLINSON&SOUTHGATE,~~~~). Throughout much of Africa, both urinary and intestinal forms of schistosomiasis occur (ROLLINSON & JOHNSTON, 1996). Urinary schistosomi&is is attributable to infection with S. haematobium whilst infection with either S. mansoni or S. intercalatum results in intestinal schistosomiasis. S. mansoni is more widespread throughout Africa than 5’. intercalatum, which has restricted distribution limited to the Democratic Republic of Congo (formerly Zaire), Equatorial Guinea, Gabon, Nigeria and Cameroon with isolated case reports from other parts of central and western Africa (BROWN et al., 1984). S. intercalatum is also found in SZo Torn& (SOUTHGATE et al., 1994). Two distinct strains of S. intercalatum are recognized, having different phenotypic characteristics and geographical distribution. S. haematobium has a wide distribution in Africa; its compatibility with species of Bulinus, the snail intermediate host, is varied and often limited to specific interactions confined to a given locality (ROLL INSON STON,
&
SOUTHGATE,
1987;
ROLLINSON
& JOHN-
1996). Other species within the S. haematobium group are of veterinary importance, including S. bovis, S. curassoni and S. mattheei; in certain areas of Africa (e.g., Sudan) S. bovis causes serious problems in livestock (ROLLINSON~LSOUTHGATE, 1987).
Address for correspondence: Mr R. A. Kane, Wolfson Wellcome Biomedical Laboratories, The Natural History Museum, Cromwell Road, London, SW7 5BD, UK; phone +44 (0)207 942 5152; fax +44 (0)207 942 5347, e-mail [email protected]
Schistosoma
intercala-
In marked contrast to the majority of Digenea, the schistosomes are dioecious and have successfully colonized the blood vasculature of vertebrates. If they are able to infect the same definitive host, there appear to be few, if any, physiological barriers preventing encounter or mating of schistosome worms of different species (SOUTHGATE et al., 1998). Hence, there are cases of hybridization in nature, especially where transmission cycles are sympatric, favouring the occurrence of mixed infections in the mammalian host. Within the S. haematobium group, several species are known to have the potential to hybridize but, interestingly, viable crosses are not always reciprocal (SOUTHGATE & ROLLINSON, 1987). Whilst hybridization between schistosome species belonging to different species groups does not occur, interspecific interactions do. For example, mixed infections of S. haematobium and S. mansoni in laboratory animals readily form heterospecific and homospecific worm pairs. S. haematobium males exhibit a greater ability to take S. mansoni females away from S. mansoni males, perhaps an indication of their greater competitiveness (WEBSTER et al., 1999). In such heterospecific pairings, identification of female worms is relatively straightforward by the examination of intrauterine egg morphology or the position of the ovary. Identification of male worms, however, is more problematic and is not easily determined by morphology. The development of better molecular methods for identification and characterization of schistosome populations will alleviate logistical problems associated with current methods and provide data useful for population genetics and epidemiology. Natural hybrids can be detected and monitored by possession of intersecting sets of molecular characters. In addition, by comparison of mitochondrial deoxyribonucleic acid (mtDNA) and nuclear DNA loci, the maternal/paternal genealogy of the hybridizing species can be determined, assuming strict maternal inheritance of mtDNA and Mendelian assortment at nuclear loci. Since the development of the single-stranded conformational polymorphism (SSCP) method by ORITA et al. (1989), the technique has become widespread within the field of medical research for mutational analysis. GASSER &
R. A. KANE
S11236
ZHU (1999) have reviewed SSCP and other mutation detection techniques based on the polymerase chain reaction (PCR) as applied to parasites, concluding that the methods have advantages over more conventional DNA methods, provided they are selected with care. Previous SSCP studies have relied upon radioactive labelling or silver staining methods (HISS et al., 1994; DE LEON et al., 1998; G~SER et al., 1998; STOTHARD et al.. 1997. 1998: B~GH et al.. 1999; LIU et al., 1999). In this pap& the ðodology;s carried a stage-further, taking advantage of modern fluorescent chemistry using an ABI Prism 377TM automated sequencer. Four Schistosoma species, including 3 isolates of S. haematobium and 2 isolates of S. intercalatum, were examined to determine whether SSCP analysis can generate informative markers. Two genetic loci from the mitochondrion and nucleus were targeted. A 177 bp fragment was studied from the mitochondrial cytochrome oxidase subunit 1 (COl) gene, and a 207 bp fragment from the nuclear ribosomal spacer ITS2. Methods Generation offluorescent PCR fragments Six individual worms from each of the 4 snecies and 7 isolates were selected for study (see the Table). DNA was extracted according to the method outlined by WALKER et al. (1986) with minor modification. PCR products for CO1 were generated using Amplitaq GoldTM nolvmerase (Perkin Elmer) and the following cycling don&ions oh a HybaidTti thermal cycler: i cycle of 10 min at 94”C, 1 min at 5O”C, and 1 min at 72”C, 30 cycles of 1 min at 94”C, 1 min at 5O”C, and 1 min at 72”C, and a final cycle of 1 min at 94”C, 1 min at 50°C and 10 min at 72°C. The sequence of the forward primer, COlSSCPl, was as follows: 5’GTT CWGTGACTATGATTAT3’ and the reverse mimer. cOlSSCP2,5’CTAATGAAGAAGCRG&GC3 The forward primer was fluorescently labelled at the 5’ end with NED (yellow) and the reverse primer with HEX (green). The ITS2 fragment was amplified using a Perkin ElmerTM 9700 cycler and Amplitaq GoldTM. The cycling programme consisted of initial denaturation for 10 min at 94”C, followed by 30 cycles of 1 min at 94”C, 1 min at 58”C, and 1 min at 72°C. There was a final elongation period of 10 min at 72°C. The forward primer, ITSSSCPl, had the sequence 5’GCA TATCAACGCGGGS’ and was labelled at the 5’ end with NED, while the sequence of the reverse primer, ITSSSCP2, was 5’ACAAACCGTAGACCGAACC3’. The latter was labelled at the 5’ end with HEX. All PCR nroducts were nurified using Nucleon OCTM clean-;p kits (Amershim Life Sciencu). Electrophoretic conditions and sample preparation SSCP analysis on an ABI Prism 377TM automated sequencer was performed using a Perkin Elmer Applied Biosystems protocol, but with the following specific modifications and run conditions. Electrophoretic gel. Non-denaturing 5% Long RangerTM gel prepared as follows: 1.25 g glycerol, 8.1 mL Table.
Origins
of isolates
Species
Country
S. S. S. S. S. S. S.
Senegal Mali Mauritius SBo Tom& Cameroon Senegal Senegal
haematobium haematobium haematobium intercalatum intercalatum curassoni bovis
used in this study NHM isolate number 3572 3125 3135 2758 1970 2517 B2
Males Females G 3 3 3 6
3 3 3 3 6 -
ETAL.
50% Long Ranger gel solution concentrate (FMC), and 30 mL distilled water. The volume was adjusted to 45 mL with distilled water, and the solution was filtered and added to 5 mL of filtered 10X tris-borate-ethylenediaminetetraacetic acid (TBE) buffer at pH 8.3. The whole was then degassed for 5 min and 250 & of 10% ammonium persulphate and 35 & of N,N,N’,N’-tetramethylethylenediamine (TEMED) were added. The resultant solution was mixed and poured into the assembled gel plates. Pre-run module on 377 DNA Sequencer. Electrophoresis voltage 1000 V. current 35 mA. Dower 50 W, nel tempera&e 20°C (iemperatures were-maintained -wyt.h an external Multitemp IIITM thermostatic circulator [Pharmacia Biotech]). The gel was pre-run for 5 min. Run module on 377 DNA Sequencer. Electrophoresis voltage 2140 V, current 60 mA, power 200 W, gel temperature 20°C. The data collection time was 13 h. Sample preparation. A 60X mix consisting of 120 ti of dei&&d~formamide containing Blue Dextran and 2.1 uL of 1 M NaOH. 2 UL of this mix were added to 1 & of appropriately d&ted PCR product and 0.75 pL of GS2500 internal lane standard (Perkin Elmer), giving a total sample volume of 3.75 &. These samples were then heated at 95°C for 5 min and placed immediately on ice. 2 & of each sample were loaded and run into the gel for 3 min using the pre-run settings before this was terminated and the full run module settings implemented. Internal lane standards were run within each sample and, because the standards were denatured together with the experimental DNA, measurement of mobility in terms of exact nucleotide length was not possible. Mobility values were therefore derived by the use of scan numbers, which are recorded by the sequencer throughout the run. All DNA sequences obtained in this study were ‘blasted’ (ALTSCHUL et al., 1990) against the EMBL database in order to ensure that they were of schistosomal origin. Results Electronic images of typical SSCP profiles for CO1 from individual worms are shown in Fig. 1A. The coloured banding patterns correspond to single stranded DNA fragments that possess either green or yellow fluorescent primers. It is immediately apparent that the reverse green strand is travelling in advance of the complementary yellow forward strand for all the species and isolates used in the study. The SSCP profile could not differentiate S. haematobium isolates from Senegal and Mali, and subsequently their DNA sequences were shown to be identical. There was, however, a notable difference between the profile obtained with S. haematobium from Mauritius and those from Senegal and Mali. Sequencing the CO1 fragment revealed that the difference in mobility was presumably att’ributed to a single substitution of thymidine (T) for cytidine (C) located 38 bp from its 3’ end and 16 bp upstream of the COlSSCP2 priming site (see Fig. 1B). This was a synonymous substitution, the amino acid, glycine, remaining unaltered. The chroinatographic profiles of the faster migrating green strand in all cases indicated a doublet, whilst the slower migrating yellow strand was shown to be a single peak. The only exceptions were the Mauritian isolate- (a triplet), and thk 4 slower mizrants of S. bovis (doublets) (see Fia. 2). In the la&, the two faster moving s&an&, in &a&s 38 and 41, differed in sequence from their neighbours by the substitution of a C for a T, 7 bp upstream from the primer COlSSCP2 and only 9 bp downstream from the similar substitution in the Mauritian S. haematobium isolate. Again, another synonymous mutation was detected, the amino acid threonine remaining un-
SSCP
ANALYSIS
OF SCHISTOSOMA
SW.
9300
9500
2517
9600
37##
CO1 chromatograms
9400 9800
4900
1QQQQ
1
ITS2 chromatograms
10200
1#4##
10600
10800
Fig. 2. Selection of chromatograms showing peak structures for the various species of Schistosoma and variable isolates. Where one isolate within a species differs in format from another, both forms are shown. In the case of S. bovis, the 2 variant forms within isolate B2 are overlapped. The aberrant S. intercalatum sample is identified by the superscript number 27 next to the NHM coding. Yellow peaks are shown in black for visual clarity. The isolate code numbers are explained in the Table. Consult the scan reading at the top of each chromatographic ‘snapshot’ to compare mobility values as the frames are not all at the same scale.
10 9200
####
$
3 q
SSCP
ANALYSIS
OF SCHISTOSOMA
SW.
S11239
II
*
.
-
4 ii
. . *
.
. . .
s11240
changed. Sample number 27, an S. intercalatum isolate from Cameroon, had an extra fragment in both green and yellow strands (see Fig. 1A). The chromatogram revealed a ‘marginal’ triplet for the reverse strand and a doublet for the forward strand (see Fig. 2). When sequenced, however, the CO1 fragment from this worm proved to be the same as that of the S. intercalaturn isolate from S5o Tome. It is therefore not certain whether the additional peaks observed were genuinely different conformations or, perhaps, an artefact. The area of a chromatographic peak may be used to determine the relative quantity of single stranded DNA present in a particular band. This area can vary slightly from track to track depending on the original concentration of DNA loaded; however, the overall proportion should remain relatively the same. Thus, using the reverse strand for the 6 individual worms of S. bovis as an example, a relative value for the percentage of single stranded molecules adopting a particular conformation can be obtained. In this case an average value for the 6 worms was calculated, revealing that 20% of the molecules adopted the structure associated with the smaller fast-moving peak, while 80% arranged themselves in a manner corresponding to the larger peak. The electronic image of the ITS2 fragment generated from the same worms revealed fewer variants and showed a completely different profile to that of CO 1, as might be expected. In this case, the forward and reverse strands had changed positions according to the species studied. All the S. haematobium PCR fragments had faster migrating yellow forward strands, while in the remaining species this strand migrated more slowly than the green counterpart (see Fig. 3A). Sequencing showed that the difference in mobility between S. haematobium and the other 3 species was due to substitutions at 3 sites. These were located as follows from the 5’ end of the fragment: position 25, where in S. haematobium adenosine (A) is replaced bv_ -guanosine (G); position 80, where T changes to C; and position 130. where A is anain substituted bv G (see Fig. 3B). In all &her positions, the sequences here‘ the sime. ‘The location of nucleotide variants towards the end regions of either the CO1 or ITS2 fragments did not appear to inhibit their detection by this method. Discussion
SSCP analysis has been based on the premise that single-stranded DNA molecules adopt unique secondary structural conformations when electrophoresed under non-denaturing conditions (ORITA et al., 1989; HISS et al., 1994; STOTHARJIet al., 1997; GASSER& ZHU, 1999). The exact nature of a particular conformation is determined by the single strand length, nucleotide sequence, and location of intra-strand pairing. Recent evidence has also shown that tertiary structures (i.e., sugar-base and sugar-sugar interactibns) are an additional determinant of mobility (LIU et al., 1999). The sensitivity of the technique is impressive, for example single point mutations w&-e detectable &thin CO1 between isolates of S. haematobium. Similarly for ITS2, where the striking difference between isolates of S. haematobium and the remaining schistosome species was attributable to just 3 point mutations. Sensitive molecular methods are needed to monitor tb.e genetic epidemiology of schistosome populations (BO*LES et al., 1993,-i995; DEW&S et-al, 1992). New techniaues for identification of moruholoeicallv similar cercaiiae would be particularly val
R. A. KANE ETAL.
intercalatum, S. cursassoni and S. bovis, and the clear differentiation of these species from S. haematobium, support previous phylogenetic work that has shown a closer evolutionary affinity among S. intercalatum, S. curassoni and S. bobis thanbetweenthese species and S. (see KANE & ROLLINSON,1994; ROLLINSON et al., 19971. Molecular methods that enable haematobium
identification of hybrid strains are particularly important. Introgression of genes from one species into another could potentially facilitate transfer of resistance factors from control-selected populations into naive populations or vice versa, or even alter intermediate host compatibility if this trait was under genetic control. Whilst previous studies employing SSCP analysis have used conventional electrophoretic apparatus and methods of detection, greater resolution and more information are obtainable through the utilization of an ABI Prism 377TM automated sequencer. As each PCR primer can be labelled with a separate fluorescent dye, both positive and negative strands can be simuitaneouslv identified and analvsed with GenescanTM and Geno&erTM software. Labilling the two DNA strands with different colours ensures that the same strands are compared across samples, thereby removing false comuarisons. The SSCP nrofiles of ITS2 are a g.ood example, where mobility 0; positive and negativeitrands can be highly divergent; without the use of 2 colours, the banding patterns could be confused. The display of both fluorescent colours together also indicates that a fragment is comprised of both strands. In addition, colour labelling permits multiplexing of different PCR products into a single lane, which can reduce both processing time and cost. Manipulation and storage of mobility data are greatly simplified with GenescanTM and GenotyperTM software. The chromatograms generated allow greater resolution between samples, minimizing the potential errors of visual interpretation, and the use of internal lane mobility standards enables adjustment between lanes. Acknowled!xements
We thank-Mr Stephen L. Hughes of the Veterinary Laboratory Agency for his assistance with GenotvpeP. We congratulate Dbuglas Barker on his long career-and contribution to trooical medicine. and wish him everv hauoiness during his retirement. Thanks* are due to the &rop,an Comm&ity (award ICI S-C196-0041) and the Wellcome Trust for finahcially supporting the work. References
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