Echinococcus multilocularis: Microsatellite ... - Science Direct

JOBNAME: JEP 82#3 96 PAGE: 1 SESS: 14 OUTPUT: Wed Jun 5 02:49:15 1996 /xypage/worksmart/tsp000/69001j/12

EXPERIMENTAL PARASITOLOGY ARTICLE NO. 0040

82, 324–328 (1996)

RESEARCH BRIEF Echinococcus multilocularis: Microsatellite Polymorphism in U1 snRNA Genes STE´ PHANE BRETAGNE,*,1 BRIGITTE ASSOULINE,† DOMINIQUE VIDAUD,† RENE´ HOUIN,* MICHEL VIDAUD†

AND

*Laboratoire de Parasitologie, Faculté de Médecine, Université de Paris XII, 8 avenue du Général Sarrail, 94010, Créteil Cedex, France; and †Laboratoire de Génétique Moléculaire, URA CNRS 1484, Faculté de Pharmacie, 4 avenue de l’Observatoire, 75006, Paris, France BRETAGNE, S., ASSOULINE, B., VIDAUD, D., HOUIN, R., AND VIDAUD, M. 1996. Echinococcus multilocularis: Microsatellite polymorphism in U1 snRNA genes. Experimental Parasitology 82, 324–328. © 1996 Academic Press, Inc. INDEX DESCRIPTORS AND ABBREVIATIONS: Echinococcus multilocularis; U1 snRNA, U1 small nuclear ribonucleic acid; PCR, polymerase chain reaction; microsatellite; base pairs (bp); ribosomal RNA (rRNA); deoxyribonucleic acid (DNA); Tris(hydroxymethyl)aminomethane (Tris); Tris-borateethylendinitrilotetraacetic acid (TBE). The ability to distinguish between strains of Echinococcus multilocularis, the causal agent of alveolar echinococcosis, has many implications for epidemiological studies (Thompson and Lymbery 1988). Strains within a species may differ in traits important for diagnosis and treatment of alveolar echinococcosis as well as in traits which influence transmission patterns. For this purpose, it is important to obtain markers to characterize populations rather than to characterize each isolate individually. A wide spectrum of techniques have been used to characterize putative E. multilocularis strains including morphological methods (Rausch 1967), infectivity trials (Bartel et al. 1992; Liance et al. 1990), antigenic variability (Gottstein 1991), and prepatent period in carnivores (Thompson and Eckert 1983). Phenotypic differences between E. multilocularis isolates were frequently observed but none could be used to define populations. Only the forms from Europe and Alaska might be considered as strains rather than subspecies (Eckert and Thompson 1988), based on morphological analyses (Rausch 1967). DNA technology has also been used to distinguish different E. multilocularis isolates. Using an anonymous DNA probe, restriction fragment length polymorphisms have been detected within a set of 10 E. multilocularis isolates (Vogel et al. 1991). These polymorphisms can discriminate some isolates from the others but no epidemiological conclusion has been drawn from this observation. More recently, sequencing of two mitochondrial genes showed little variation between E. multilocularis isolates. Only 4 isolates were studied: the European isolate

1 To whom correspondence should be addressed at Laboratoire de Parasitologie, Faculté de Médecine, 8 avenue du Général Sarrail, 94010, Créteil, Cedex, France.

was different from the 3 North Americans isolates (Bowles et al. 1992; Bowles and McManus 1993). The rRNA genes have been used extensively for phylogenetic analyses of several parasites due to their accessibility and their tandemly repeated structure with spacers between each transcription unit. Most studies have focused on distinguishing between species (Nadler 1990) but few on studying sibling species (Porter and Collins 1991) or populations (De Merida et al. 1995). The E. multilocularis U1 snRNA genes are localized in one cluster and show an organization similar to that of rRNA. The gene repeat unit is 1300 bp long, more than 50 times tandemly repeated, and consists of spacers and a transcribed region of 156 bp (Bretagne et al. 1991). To validate this, we sequenced two different clones containing the 1300-bp repeat and differences in the spacers were observed. These differences consisted of a variable number of microsatellites of three, four, or five nucleotides. Microsatellites, tested by PCR, are currently largely used for analyses in human genetics (Weissenbach et al. 1992). Moreover, this technique is more reliable for comparisons between laboratories than random amplification of polymorphic DNA, largely used for typing parasites. We have, therefore, investigated whether the microsatellites in the U1 snRNA genes of E. multilocularis could be used as genetic markers. Forty-one E. multilocularis isolates were studied (Table I). Metacestodes were established and subsequently maintained in jirds (Meriones unguiculatus) by serial intraperitoneal passages and stored in liquid nitrogen since 1986 (Bretagne et al. 1990). Cysts of 11 isolates were sent to us in 70% alcohol by other laboratories. Thirty isolates were obtained from Europe, 8 from North America, and 3 from Japan. Twenty-two isolates were originally obtained from human patients, 17 from rodents, and 2 from foxes. High-

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MICROSATELLITES OF

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TABLE I Geographical Origin and Host of E. multilocularis Isolates Tested with the Results of the Profiles Obtained upon Amplification

Isolate

Geographical origin (French department)

Initial host

Date obtained

PCR profile

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37b 38 39 40 41

East France (25) East France (25) Stuttgart, Germany East France (25) East France (25) East France (25) East France (25) Alaska, U.S.A. Switzerland East France (25) East France (25) Alaska, U.S.A. East France (70) East France (39) Hokkaido, Japan East France (70) East France (25) East France (70) East France (25) East France (54) East France (25) East France (1) East France (25) East France (25) East France (25) East France (54) Alaska, U.S.A. East France (54) East France (25) East France (54) East France (54) East France (54) Hokkaido, Japan Hokkaido, Japan South East France (74) Central France (15) Alaska, U.S.A. Alaska, U.S.A. Alaska, U.S.A. Alaska, U.S.A. Montana, U.S.A.

Microtus arvalis Microtus arvalis Ondatra zibethicus Patient Microtus arvalis Patient Arvicola terrestris Sigmodon hispidusa Patient Patient Patient Sigmodon hispidusa Patient Patient Clethrionomys rufocanus Patient Patient Patient Patient Microtus arvalis Patient Patient Patient Patient Patient Microtus arvalis Microtus oeconomus Microtus arvalis Patient Microtus arvalis Microtus arvalis Microtus arvalis Patient Patient Vulpes vulpes Vulpes vulpes Microtus oeconomus Patient Patient Patient Rodent

1987 1984 1984 1986 1987 1988 1979 1983 1981 1986 1988 1982 1988 1987 1983 1987 1987 1986 1988 1988 1988 1988 1987 1989 1989 1989 1989 1990 1989 1990 1990 1990 1993 1993 1993 1994 1994 1994 1994 1994 1994

A A A A A A A B A A A A A A B A A A A A A A A A A A C A A A A A B B A A C B C B C

a b

Initial host at the arrival in our laboratory. Isolate identical to isolate 27.

molecular-weight DNA was extracted from cysts as previously described (Yap and Thompson 1987) as well as human and rodent DNAs. These latter were used as controls as E. multilocularis cysts are always contaminated with host cells and controls are required to assess the specificity of amplification.

The primers were designed to amplify the pentameric microsatellite upstream from the coding region of the U1 snRNA gene (Accession No. from EMBL/GenBank: M73768). The following primers were used: (sense) 59GACATTGTCGTTGCCATCTCTCCCA-39 (nucleotides 42–66), located 379 bp upstream from the 59 end of the


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transcribed region, and (antisense) 59-TTAGAGTCCGTCGCAGGCTTCAAC-39 (nucleotides 439–462), located within the transcribed region. The upper primer was labeled at the 59 end with fluorescein (Genset, Paris, France). The reaction mixture consisted of 100 ng of E. multilocularis DNA, 10 mM Tris–HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.01% (w/v) gelatin, 0.2 mM each of dNTP, 20 pmol of primers, and 1 unit of Thermophilus aquaticus 1 polymerase (Perkin–Elmer Cetus, Paris, France) in a 100-ml reaction volume. All the amplifications were carried out using a Perkin–Elmer Cetus DNA thermal cycler and included 30 cycles of denaturation at 94°C, annealing at 55°C, and extension at 72°C for 1 min each. The final cycle was followed by an additional 7 min at 72°C to complete partial polymerization. PCR products were diluted 1/10 in water and 1 ml of these was run on a 24-cm-long acrylamide urea gel (6% acrylamide, 8.3 M urea, 1 × TBE) for 8 hr under 1500 V. Signals were read using an automatic sequencer (Applied Biosystems) with an internal standard of l phage DNA digested with PstI and labeled with 6-carboxyX-rhodamine dye. The denaturing gel prevented heteroduplex formation, and, thus, only single strands were analyzed. Moreover, as each single strand contained one labeled molecule, the laser detection allowed product quantification from the peak area. The data were stored and analyzed with the 372 Genescan software (Applied Biosystems). Carryover was prevented by ultraviolet irradiation of the PCR mix (Sarkar and Sommer 1990) and by performing DNA extraction and amplification in two separate laboratories. Three different profiles, called A, B, and C, were observed (Fig. 1) and the results are listed in Table I. Amplification was specific for E. multilocularis DNA, since no amplification occurred with rodent or human DNAs. Profile A was found for 30 European isolates and was characterized by the presence of an identifiable peak at 414 bp and by the ratio of the peak area at 421 bp over the peak area at 429 bp always $2.5. Profile B was found for 3 isolates from Alaska (isolates 8, 38, and 40) and 3 from Japan (isolates 15, 33, and 34) and was characterized by the absence of any identifiable peak at 414 bp and a ratio of the peak area at 421 bp

over the peak area at 429 always #0.5. Profile C, found for 3 isolates from Alaska (isolates 27, 37, and 39) and 1 from Montana (isolate 41), did not show either previous profile but was characterized by longer PCR products, from 411 to 439 bp (Fig. 1). Isolate 12 (Table I) had a pattern similar to the European profile, although it was obtained in 1982 from a North American laboratory and its origin is uncertain. This isolate had been maintained in cotton rats (Sigmondon hispidus) and had lost its capacity to form protoscolesces, probably due to the numerous passages through rodents. It was cryopreserved only in 1987. This finding raises the issue of a misidentification during the subcultures. The same results were repeatedly observed with the same DNAs and with two DNA samples extracted from the same isolate. The results did not depend on the number of amplification cycles as they were similar after 20 cycles instead of 30; only the quantity of amplified products was weaker. Moreover, isolate 37 was shown to be identical to isolate 27 and came from the same rodent. This isolate was maintained in two different laboratories for more than 5 years and kept the same profile. Therefore, the profiles seem stable upon subcultures. To confirm that the differences between the peaks were due to a different number of pentameric microsatellites, three PCR products, one for each profile, were cloned into the plasmid T7 blue (TA cloning, Novagen, WI) and four clones of each transformation were sequenced using the 373 DNA Sequencer (Applied Biosystems) and the Taq Dye Deoxy Terminator cycle sequencing kit (Applied-Biosystems, Paris, France). Differences in the number but also in the sequences of pentamers were observed (Table II). This was equally observed in mammalian microsatellites where in addition to variation in repeat unit copy number, the precise sequence of the tandemly repeated unit showed variation within the array (Jeffreys et al. 1991). Other differences in the sequences of other PCR fragments could not be excluded but, in practice, observation of length changes remains the most convenient method for ascertainment of mutations at microsatellite loci. Thus, the spacers present in the U1 snRNA gene cluster vary in sequence, length, and

TABLE II Microsatellite Polymorphisms between Three Geographically Different E. multilocularis Isolates Isolate

Number of clones

PCR product length (bp)

Microsatellite sequences

7

2 2 1 2 1 2 2

401 411 401 411 416 416 426

GCGAG (GCAAG)2 GCGAG GCGAG (GCAAG)4 GCGAG (GCGAG)4 GCGAG (GCAAG)4 GCGAG GCGAG (GCAAG)5 GCGAG (GCGAG)4 (GCAAG)2 GCGAG (GCGAG)6 (GCAAG)2 GCGAG

15

27

Note. The PCR products were cloned into plasmid and four clones of each isolate were sequenced. The size of the PCR products refers to the size in Fig. 1 and the name of the isolates to Table I.


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relative abundance for a given isolate, mainly, if not exclusively, due to differences in pentameric microsatellites. During amplification, these different spacers are amplified according to their number at the first step of the amplification (Ruano and Kidd 1992). For rRNA spacers, a mutation from one spacer sequence spreads to the neighboring spacers faster than new changes arise in the sequences. This phenomenon was called concerted evolution and a large body of data has attested to the generality of concerted evolution in multigene families. Although there is no publication on the evolution of spacers of U1 snRNA genes, it seems likely that their evolution has followed the same way as rRNA genes. A number of mechanisms may currently be proposed to account for length-change mutations including replication slippage, unequal sister chromatid exchange, and unequal recombination between alleles (Armour et al. 1993). From the evolutionary point of view, there is an analogy between the evolution of multigene families and the evolution of subdivided populations. Therefore, the polymorphism observed in the distribution of the microsattellites within the U1 snRNA gene complexes is in keeping with the geographical separation of the parasite isolates. Thus, our results indicate that the European and the North American foci could be due to different strains, strengthening previous conclusions (Rausch 1967) and recent findings (Bowles et al. 1992; Bowles and McManus 1993). These results also confirm that the Japanese E. multilocularis focus is related to the Alaskan focus as a consequence of the importation of American foxes to Hokkaido island in the 1960s (Schantz et al. 1991). Microsatellite studies represent a new tool for addressing some epidemiological questions in parasitology. Other E. multilocularis microsatellites, presently unknown, might be used as genetic markers for drug susceptibility or other phenotypic characteristics and should be looked for. Moreover, the use of an automatic sequencer should provide the possibility to obtain reproducible data easily comparable between laboratories.

ACKNOWLEDGMENTS The authors thank Professors J. L. Rausch, T. Duriez, A. F. Pétavy, D. Vuitton, D. E. Worley, and K. Furuya for providing E. multilocularis isolates and Dr. B. Robert for helpful discussion.

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Received 6 November 1995; accepted with revision 27 February 1996