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Echinococcus granulosus strain typing in North Africa: comparison of eight nuclear and mitochondrial DNA fragments
Published online by Cambridge University Press: 01 March 2004
Abstract
Recent studies of Echinococcus granulosus molecular strain typing have enabled a better understanding of the transmission cycle of cystic echinococcosis. There have been many publications in this area but there is a need for the evaluation of these tools. We have attempted to respond to this need in our study, which assessed 8 DNA fragments of 40 E. granulosus cysts from North Africa. Parasitological material was collected from 5 types of intermediate hosts, in 5 different countries. The primers chosen to amplify DNA targets were defined either in nuclear DNA, or in mitochondrial DNA. After amplification, PCR products were sequenced. The sequences obtained were aligned and comparisons were made within the group and with GenBank sequences. Whether the target was nuclear or mitochondrial, the same 2 main groups of genotypes were found. The first one, the ‘sheep’ strain, was found in the human, sheep and cattle samples collected in North Africa. The second one, the ‘camel’ strain, was found in the camel cysts and cattle and human cysts from Mauritania. These findings further confirm the congruence of the data given by the nuclear and the mitochondrial genome.
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- 2004 Cambridge University Press
INTRODUCTION
Different methods have been proposed, including morphological description, isoenzyme analysis and genomic studies, to characterize Echinococcus granulosus (E. granulosus) strains. Morphological description has proved unreliable for identification of strains because morphological differences are host-influenced (Lymbery, Thompson & Hobbs, 1990). Isoenzyme characterization methods have proved efficient in identifying intraspecific and intrageneric variations (Macpherson & McManus, 1982) but a major disadvantage is that large quantities of pure parasitic material are required. In addition, Bryant & Flockhart (1986) have demonstrated that variations in enzyme expression may reflect regulatory changes induced by environmental influences, rather than genetic difference. The most recent approaches, genomically based, focus on DNA rather than gene products. Molecular typing methods avoid problems posed by life-cycle stage variations and environmentally- and host-induced modifications (McManus & Bowles, 1996). Comparison of PCR-amplified DNA sequences appears to be the most reproducible and the most discriminating approach (Le, Blair & McManus, 2000). Detecting nucleotide-level variations provides comprehensive information of the fragment studied. In addition, the sequences obtained in different laboratories can be easily compared thanks to databases available on Internet. Several papers have been published on sequencing of PCR products as a tool for characterization of strains and reconstitution of their life-cycle (Bowles, Blair & McManus, 1992; Haag et al. 1999; Snabel et al. 2000; van Herwerden, Gasser & Blair, 2000; Zhang, Joshi & McManus, 2000). These authors focused on mitochondrial or nuclear DNA, coding or not coding, repeated or non-repeated sequences.
The objectives of the present study were therefore to evaluate 8 DNA targets (6 nuclear and 2 mitochondrial) on a panel of cysts collected in North Africa, a cystic echinococcosis (CE) focus about which little is known (Macpherson & Wachira, 1997). The respective advantages and drawbacks of the targets were analysed in terms of sensitivity, discriminatory power and ease of interpreting results.
MATERIALS AND METHODS
Parasites
A total of 37 hydatid cysts was collected by local veterinary teams: 25 Algerian lung cysts from 10 cattle, 5 humans, 5 sheep (slaughtered in the Constantine area) and 5 camels (slaughtered in Touggourt), 12 Mauritanian lung cysts from 10 camels, 1 human and 1 bovine (slaughtered in Nouakchott). All cysts were declared fertile (presence of protoscoleces) by veterinarians following a microscopic check. Samples contained few or no bacteria.
Three additional E. granulosus DNA samples (1 Egyptian sheep, 1 Ethiopian horse and 1 Swiss bovine) completed the panel.
DNA extraction
Genomic DNA was extracted from protoscoleces obtained from individual hydatid cysts using the High Pure PCR Template Preparation kit (Roche Diagnostics, Mannheim, Germany) based on Proteinase K digestion. The DNA obtained was used either immediately for PCR amplification or stored at 4 °C until used.
Molecular analysis
Eight targets defined from the available published articles on molecular biology of Echinococcus sp. were evaluated. Their description is as follows. (i) NDI is a region of the mitochondrial NADH dehydrogenase I gene (Bowles et al. 1992). (ii) COI is a portion of the mitochondrial gene which codes for cytochrome c oxydase subunit 1. Due to these 2 targets, 9 different genotypes within E. granulosus have been described (G1 to G9). (iii) AgB/1 is a gene which codes for a protein with elastase and neutrophil chemotaxis inhibition activity. It has been used to characterize human, cattle and camel cysts (Frosch et al. 1994). (iv) EmHbX2 is an intron from a homeogene involved in E. granulosus development regulation (Haag et al. 1997). (v) ActII is defined as an intron extract from the gene EgActII (da Silva et al. 1993). (vi) BG 1/3 is a repeated DNA fragment identified within the E. multilocularis genome (Gottstein & Mowatt, 1991). (vii) ITS-1 is the first internal transcribed spacer, in a non-coding region of ribosomal DNA (van Herwerden et al. 2000). (viii) MS is a microsatellite identified within the U1 snRNA genes of E. multilocularis (Bretagne et al. 1996).
Eleven pairs of primers were designed to amplify these 8 targets. The PCR programs contained 40 cycles with, for each cycle, a denaturation step (30s at 94 °C), a hybridization step (30–75s between 45 to 60 °C according to the primer characteristics) and an elongation step (30s at 72 °C) (Table 1). For each PCR reaction, 4 μl of genomic DNA were mixed with 5 μl PCR Buffer 10X (Sigma™, St Louis, MO, USA), 20 μM dNTP (dNTPset, MBI Fermentas, Vilnius, Lithuania), 40 pmol of each primer (Invitrogen Life Technologies™, Paisley, Scotland) and 1.25 U Taq polymerase (REDTaq-Polymerase™). The final volume was 50 μl. After confirmation of the amplification in a 2% agarose gel, 5 μl of each amplified fragment were purified using 2 μl EXOSAP-IT enzyme (Amersham Pharmacia Biotech Europe, Orsay, France). The purified DNA was then sequenced using of the DYEnamic-ET terminator cycle sequencing kit (Amersham Pharmacia Biotech Europe GmbH, Freiberg, Germany). Fragment detection was performed by an automated DNA sequencer 373A (Perkin Elmer Applied Biosystems, Foster City, CA, USA). Reverse DNA strands were systematically sequenced for each target. Sequenced data were afterwards aligned using CLUSTALW software (http://bioweb.pasteur.fr) and compared to those previously published in the Genbank data base (http://www.ncbi.nlm.nih.gov) with the BLAST system.
Criteria for evaluation of DNA targets
The relevance of the data obtained for the 8 targets was evaluated according to 5 criteria. (1) PCR-amplification sensitivity (capacity of a target to be amplified for all the samples), (2) reproducibility (consistency of the results found by experimenters), (3) repeatability (consistency of the results repeated by the same experimenter), (4) the facility in interpretation of the sequences obtained, and (5) the polymorphism of these sequences (mutation rate between the different samples).
RESULTS
PCR results
The 2 mtDNA fragments (COI and NDI) were amplified for all the panel samples, in agreement with the published data.
AgB/1 and ActII 1/2 yielded no PCR product. The ActK 1/2 primers, defined according to the ActII gene sequence published in GenBank, gave an amplification of a 0·4 kb fragment for the 40 samples. With the ITS-1 target, BD1/4S primers amplified not only a 1 kb PCR product as previously published, but also 2 additional 0·2 and 0·3 kb products. We designed a smaller target (theoretical size: 0·3 kb) in ITS-1 (with the ITSB 1/2 primers), which produced amplification with the Mauritanian and the Algerian camel samples but not with the other panel samples. MSK 1/4 primers produced amplification of all panel samples. A size polymorphism differentiated 2 groups: the human cysts (Algerian, Moroccan and Egyptian) and the Algerian cattle and sheep cysts presented with a 190 bp band, whereas the Mauritanian and Algerian camel cysts presented with a 175 bp band. The HbX 1/2 primers amplified the EmHbX2 target for all the samples. The last nuclear target, BG1/3, showed 2 bands, the expected 300 bp band and a smaller one at 165 bp, for the Mauritanian and Algerian camel samples, and only one, the expected 300 bp, for the other samples.
Sequencing results
Mitochondrial targets.
The highest rate of polymorphism was found for the mitochondrial targets with 41–0 differences/366 bp for COI and 60–0 differences/481 bp for NDI. According to the sequences of the mitochondrial genes available in GenBank, the Algerian samples from human, ovine and cattle cysts as well as the Moroccan and the Egyptian samples had a genotype with a close resemblance to G1, the ‘sheep strain’ (Genbank Reference: AF297617). The ‘micro-variants’ observed had a maximum of 3 divergent bases in comparison with the reference sequence. For 3 samples (2 human and 1 ovine) amplified with the NDI target, the electrophoregrams showed some mixed peaks. On each of these peaks, nucleotides characteristic of G1 and G6 were superposed. The Mauritanian and the Algerian camel samples presented a G6 profile, characteristic of the ‘camel strain’. The Ethiopian horse and the Swiss cattle samples belonged respectively to G4 and the G5 genotypes (GbR: AF346403 and AJ237636 respectively).
Nuclear targets.
Microsatellite MS detected 4 distinct genotypic groups. The first one, characterized by the pattern (gACgA)4 and (ggCgA)4 repetitions, was composed of the Algerian and Moroccan human, the Algerian and Egyptian sheep and the Algerian cattle samples. The second one, composed of the Mauritanian samples and the Algerian camel, was (gACgA)4 and (ggCgA)1. The genotype of the Ethiopian horse was (gACgA)1 and (ggCgA)9 and that of the Swiss bovine was (gACgA)6 and (ggCgA)1. Using BG1/3 and EmHbX2, homology between all samples varied between 98·3 and 100% (GbR: AY129567 and AY129568). With these targets, the 4 genotypic groups described above were still differentiated. However, within the first group, the Egyptian sheep sample could be distinguished from human and ovine samples collected in Maghreb, whereas the sequences found in Algerian human, sheep and cattle samples and in the Moroccan human samples were identical. Lastly, the ActK1/2 target showed a high level of polymorphism, with 96·4 to 100% homology (15–0 differences/420 bp) between the samples (GbR: AF528498, AF528499, AF528500). Strain typing by sequencing was found very reproducible for all targets studied; repeated testing performed in at least 3 samples for each sequenced target gave identical data (Table 2).
DISCUSSION
Given the various molecular tools (6 nuclear and 2 mitochondrial targets) to characterize our 40 samples, the results obtained in our study were very congruent. Both mitochondrial and nuclear targets defined the same 4 epidemiological groups: the first group composed of human, ovine and cattle samples collected in North Africa; the second group of camel cysts, whatever their source, as well as cattle and human cysts from Mauritania; the third group, of an Ethiopian horse; and the fourth group, of a Swiss bovine. These groups, respectively G1, G6, G4 and G5 genotypes, were previously described within the genus Echinococcus.
The same major groups of genotypes were found using both of the mitochondrial targets, despite their degrees of polymorphism within a strain. In fact, the major advantage of using mitochondrial targets is that data can be compared with the many other sequences found worldwide which are published in Genbank. As shown by the data published on the properties of the mtDNA (Vawter & Brown, 1986), the mitochondrial targets (COI and NDI) were the most polymorphic. The additional micro-variants we found using sequences present in the mitochondrial genome did not seem to present any epidemiological relevance, at least at the scale we worked, i.e. in defined areas of a continent. Kamenetzky et al. (2002) in a recent study performed in Argentina, noticed the same type of micro-variations within the ‘sheep’ strain using COI sequences for typing.
The ambiguous patterns obtained for 3 NDI sequences have already been described (Bowles, Blair & McManus, 1994). In addition, with this target, some difficulties appeared for sequencing the G1 strain samples. New primers, adapted to the E. granulosus mitochondrial genome, need to be designed in order to facilitate interpretation of this particular DNA fragment.
However, mitochondrial targets do have some drawbacks. First, they focus only on the portion of the genome that has a particular mode of transmission from one generation to the next. Contrary to the nuclear genome, mitochondrial DNA is generally assumed to be uniparentally inherited (except in rare cases). In other words, it is accepted that the different lineages of mitochondria do not recombine. Studies have shown that breeding outcrossing could occur in E. granulosus (Haag et al. 1998, 1999). Consequently, by using only mitochondrial targets in typing strains, there is, at least theoretically, the risk that important information on genetic modifications between individual parasites could be lost. Other authors have already found that the use of NDI and COI alone is insufficient for phylogenetic analysis (Okamoto et al. 1995; Thompson & Lymbery, 1990). They have also proposed the characterization of nuclear DNA fragments such as rDNA internal transcribed spacer region.
It must be recognized that all the targets we studied have their limitations for use in ‘molecular epidemiology’ (Tibayrenc, 1998) studies of Echinococcus. The genotypes which we obtained, either with mitochondrial or nuclear targets, still failed to identify, on a small scale, particularities within a strain. For example, we could not differentiate the Algerian camel E. granulosus strains from the Mauritanian ones with any of the targets studied, even though the cysts had been collected in slaughterhouses separated by thousands of kilometres of desert. The same problem occurred in identifying geographical differences within human and ovine samples collected in Morocco and in the North of Algeria. Discovery of more polymorphic genetic markers would be of great importance in the study of the host–parasite relationship, population genetics, or the influence of geographical conditions on the evolution of the genome.
We are grateful to Lois Rose for her editorial assistance.
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Table 1. Primer sequences and PCR conditions for amplification of the targets
Table 2. Evaluation of targets used for E. granulosus strain typing
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