Rev. sci. tech. Off. int. Epiz., 1990, 9 (2), 489-510

Characterisation of taeniid cestodes by DNA analysis

D.P. McMANUS *

Summary: This review describes how the application of molecular technology is providing improved methods for characterising and identifying taeniid cestodes. Emphasis has been placed on the use of DNA approaches for specific diagnosis of the various taeniid life-cycle stages, unambiguous species and strain discrimination, determination of evolutionary and phylogenetic relationships and the assignment of function to key macromolecules based on inferred amino acid sequences corresponding to cloned cDNA sequences. In addition, the review describes methods for isolating DNA from taeniids, approaches for cloning genomic and mitochondrial DNA from these organisms and the use of DNA methods for strain characterisation and egg detection. Lastly, the review focuses on the polymerase chain reaction (PCR) which can provide the basis for exquisitely sensitive assays diagnostic for individual taeniid species. Coupled with direct sequencing techniques, PCR can also provide a valuable new method for examining genetic variation between and within the various taeniid species.

KEYWORDS: Cloned Echinococcus cDNA - Cloning taeniid cestode DNA -Direct sequencing - DNA detection of eggs - Echinococcus - Polymerase chain reaction - Strain characterisation - Taenia - Taeniid cestodes.

INTRODUCTION

The most important cyclophyllidean cestode family from the medical or veterinary standpoint is the Taeniidae; species comprising this group are also called taeniid cestodes or taeniids. Echinococcus spp., which use canids as definitive hosts and a wide range of intermediate hosts, are the causative organisms of hydatid disease or hydatidosis in man and animals; the two most important species, E. granulosus and E. multilocularis cause unilocular and alveolar hydatid disease respectively. Cysticercosis is due to the Cysticercus (Cysticercus cellulosae) of the pig tapeworm, Taenia solium; pigs act as the intermediate host and the adult worms occur in the human gut. Man can also act as intermediate host of T. solium where the cysticerci cause a serious and often fatal disease due to the frequent involvement of the nervous system (neurocysticercosis). Other important taeniid species include Taenia saginata, which causes bovine cysticercosis (adult in man) and T. ovis (ovine cysticercosis), T. hydatigena and T. multlceps in sheep (adults in dogs). T. taeniaeformis in rats and mice, T. crassiceps in mice and T. pisiformis in rabbits have proved valuable experimental models for biological and immunological studies of cysticercosis and hydatid disease.

* Tropical Health Program, Queensland Institute of Medical Research, Bramston Terrace, Herston, Brisbane, Queensland 4006, Australia.

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The biology, physiology and biochemistry (including molecular biology) of the group have been recently reviewed (41). In addition, useful guidelines describing the surveillance, prevention and control of echinococcosis/hydatidosis (2) and taeniasis/cysticercosis (3) are available. The control of these taeniid infections depends on carefully collected epidemiological information, including precise identification and characterisation of the causative agent in each endemic area. The recent application of molecular biological technology, including the use of DNA modifying enzymes (e.g. restriction endonucleases, DNA ligases) to cut and ligate DNA, prokaryotic plasmid and bacteriophage-based vectors to clone and prepare large quantities of specific DNA sequences, DNA sequencing techniques, the use of synthetic DNA molecules and the polymerase chain reaction (PCR), has powered a revolution in the study of these and other parasites and the diseases they cause. This technology is proving of value for characterising taeniid cestodes in areas such as the specific diagnosis of the various life-cycle stages, including the egg, unambiguous species and intra-species (strain) discrimination, the assignment of function to important macromolecules, based on DNA sequence information, and in the definition of evolutionary and phylogenetic relationships.

BASIC APPROACHES FOR D N A ISOLATION

For successful DNA analysis of a particular taeniid species, the nucleic acid generally has to be prepared so that it can be cleaved by various enzymes such as restriction endonucleases. A very convenient and repeatable method, which has proved useful for the isolation of DNA from a range of taeniids, is shown in Fig. 1. Thus, high molecular weight DNA has been extracted from freshly isolated or frozen adults and protoscoleces (aspirated from hydatid cyst material) of E. granulosus and E. multilocularis (21, 26, 34) but not from hydatid fluid or the germinal/laminated layers (34). Moreover, of practical value, DNA can be isolated from Echinococcus material lyophilised or fixed (for 12 months or more) in 70% ethanol (but not formalin which degrades high molecular weight DNA) (26). This facilitates transport of parasites from slaughterhouses, operating theatres or the field situation to the laboratory for analysis. Similarly, high molecular weight DNA can be isolated from fresh or frozen larvae and adult Taenia spp. and from ethanol-fixed tissue (35, 36) although care has to be taken in the preparation of the material. Experience has shown that parasites must be processed immediately after they are obtained from faeces, the host gut or dissected from tissues. If large and to be ethanol-fixed, they should be compressed (e.g. between glass slides) and placed in ethanol to facilitate rapid fixation, thereby preventing degradation of the DNA by endogenous or exogenous enzymes. Other methods used for the isolation of taeniid DNA include caesium chloride density ultracentrifugation (21, 52) and the selective precipitation of nucleic acids by cetyltrimethylammonium bromide (50). The latter technique has also been used to extract mitochondrial DNA (mtDNA) from cestode tissue (52).

CLONING OF TAENLIID CESTODE D N A

General principles

The general principles of DNA cloning, the insertion of a piece of foreign DNA into a vector plasmid or bacteriophage and its multiplication in bacteria are well known

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Frozen parasites (1 vol. packed worms) crushed in liquid nitrogen

Powder thawed into 4 vol. extraction buffer

Equal vol. extraction buffer containing 1 % SDS then added, followed by 1 mg Proteinase K

Incubate for 3 h

Phenol extraction, phenol/chloroform extraction and chloroform extraction

Total nucleic acids in supernatant precipitated in 0.3 M sodium acetate and 2.5 vol. ethanol overnight

at - 2 0 ° C and pelleted at 10,000 g, 10 min, 0°C

Total nucleic acids dried and dissolved In 4 ml 10 mM Hepes, pH 7.5

A method for the isolation of DNA from taeniid cestodes [After McManus et al. (21)]

* As an alternative, instead of adding LiCl, RNAse can be used at this stage to digest RNA. This is followed by sequential phenol, phenol/chloroform, chloroform extractions and subsequent DNA isolation by ethanol precipitation. The DNA in the pelleted total nucleic acids fraction is sufficiently pure for many subsequent procedures.

TE buffer is 10 mM Tris-HCl (pH 8.0).

3 vol. of 4 M LiCl added and left overnight *

RNA precipitate pelleted at 10,000 g, 10 min. Washed in 70% ethanol, dried and dissolved in 0.1 ml 10 mM Hepes, pH 7.5

DNA isolated from supernatant by addition of 2.5 vol. ethanol and subsequent spooling or centrifugation at 10,000 g, 10 min, 0°C. Washed in 70% ethanol, dried and dissolved in 0.5 ml TE buffer

FIG. 1

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and will not be described here. Details of these and other general procedures in molecular biology, applicable to taeniid cestodes, are described by Sambrook, Fritsch and Maniatis (39).

The DNA of any organism is the ultimate blueprint of that organism and, in parasites, it is the one characteristic which generally remains unchanged during every life-cycle stage. Consequently, the entire DNA sequence in the egg, larva, adult, free-living and parasitic stages of a particular species are the same. DNA methods overcome the inherent problem of other identification procedures such as isoenzyme analysis or the use of monoclonal antibodies which analyse the expressed products of the genome and can show life-stage or environmentally mediated, including host-induced, variation, whereas DNA procedures do not. Several groups have used cloning strategies in attempts to isolate and purify recombinant DNA clones containing specific DNA sequences to facilitate improved identification of taeniid species and strains. This work is discussed below.

Cloning of genomic DNA sequences from Echinococcus spp.

In the first study of its type with a taeniid, Rishi and McManus (34) constructed a small, size-selected genomic DNA library in the bacterial plasmid pAT153 using total DNA extracted from a human isolate of E. granulosus. Subsequent screening of the library with a panel of DNA's from a range of taeniid cestodes, including E. granulosus and E. multilocularis, identified two recombinant plasmids containing Echinococcus-specific DNA inserts (coded pEG16 and pEG22) and one with an E. granulosus-speciiic DNA insert (coded pEG18). These and other recombinant plasmids selected from the library were subsequently used as DNA probes in strain characterisation (described below). The potential of pEG16, pEG22 and pEG18 as DNA probes in a field assay for distinguishing eggs of Echinococcus from those of other species remains to be assessed, although the low copy number ( = 27) of the insert (2.3 kb in size) of pEG18 may limit its use in the detection of small numbers of eggs without PCR amplification of the target DNA. The potential of this technology is discussed later.

A recombinant plasmid (coded pALl), specific for E. multilocularis and E. granulosus, has been produced by another group (49). This DNA probe was obtained by subcloning a 0.6 kb DNA fragment, originally isolated from a genomic phage lambda EMBL4 sublibrary of E. multilocularis, into the bacterial plasmid Bluescript M13 + (BS + ). This probe can clearly discriminate between the two Echinococcus spp. in Southern hybridisation experiments.

Cloning of genomic DNA sequences from Taenia spp.

Great clinical and epidemiological importance has been given to developing a simple test to distinguish T. solium from T. saginata. Particular requirements are the specific diagnosis of T. solium tapeworm carriers, because of the risk of either the host or his human and porcine contacts developing cysticercosis, the identification of cysticerci or cyst residues in bovine or porcine carcasses after slaughter or from human biopsy material and the specific identification of taeniid eggs on pasture or in human faecal samples. Morphological criteria are often insufficient to make an accurate diagnosis and isoenzyme techniques are somewhat impractical. Consequently, recent studies have concentrated mainly on the identification and cloning of species-specific DNA sequences and their application as DNA probes in appropriate assays. DNA probes, specific for T. solium and T. saginata, are now available.

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Two T. solium DNA sequences have been cloned in pAT153 recombinant plasmids (coded pTS1O and pTS17) using a cloning strategy similar to that described above for E. granulosus (36). pTS1O was shown to have absolute specificity for T. solium while pTS17 had selective specificity for T. solium and the dog/sheep taeniid T. ovis. Both probes hybridised to T. solium isolates from Zimbabwe, Mexico and India and could successfully discriminate T. solium from T. saginata in Southern hybridisation analysis and in the simpler dot-blot (slot-blot) assay. The approach provides a rapid and precise assay for identification of T. solium segments in hospital pathology laboratories and should prove of value for field epidemiological surveys where differential diagnosis of segments of T. solium from T. saginata is required (35). Cloned rDNA and/or 32P-labelled total RNA sequences have also been used as DNA probes in Southern hybridisation analysis to distinguish a range of taeniids including a new species of Taenia from Taiwan (36; Zarlenga, McManus, Fan and Cross, unpublished).

In another series of experiments (16; see also 5), a genomic DNA library was constructed in the bacteriophage lambda gt1O using T. saginata DNA. Differential screening of the library with a panel of taeniid DNA's identified two recombinant phages, one with a DNA insert specific for T. solium and the other containing a DNA sequence specific for T. solium and T. saginata. The combined use of these two probes allows both positive identification and discrimination of the two species (Fig. 2).

Cloning of taeniid cestode mtDNA

The mitochondrial genome of metazoa is, with one known exception, contained in a single circular molecule with a species-specific size varying from 14-39 kb. Studies, originally with closely related mammals but then with a range of organisms, have shown that mtDNA has a higher rate of evolutionary substitution (i.e. it evolves faster) than does nuclear DNA (18). Consequently, mtDNA has been shown to be of value in studies on the systematics and phylogeny of plants and animals and is useful for examining affinities and divergences among closely related groups (52). To date, very little is known about the mtDNA of taeniid cestodes although the isolation of mtDNA from several species was recently described (52). Comparative restriction analysis of the mtDNA produced uncomplicated, distinctive restriction patterns by which these species could be readily distinguished. Additionally in this study, the mtDNA of T. hydatigena was characterised and was found to be circular and similar in size (17.6 kb) to that of other animal species. Subsequently, the entire mt genome of T. hydatigena was cloned by the group into pBR322 in Escherichia coli and a restriction map of the recombinant molecule constructed (Fig. 3).

STRAIN CHARACTERISATION

There is now overwhelming evidence that many parasitic helminths, including some taeniid cestodes, exhibit substantial genetic diversity (37) and this may have important implications for the design and development of vaccines, diagnostic reagents and drugs effective against these parasites. DNA technology is providing uniquely sensitive and specific assays whereby these genetic variants can be rapidly characterised.

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1 2 3 4

Specific probe Cross-reactive probe

FIG. 2

DNA probes allowing identification and discrimination of Taenia saginata and T. solium

[After Barker (5)]

Doubling dilutions (200 ng-3 ng) of T. saginata DNA (tracks 2 and 4) and T. solium DNA (tracks 1 and 3) were blotted onto nitrocellulose and then probed with 32P-labelled bacteriophage DNA containing a T. saginata-specific insert (200 ng at 4 x 108 dpm/ng), exposed for 2 days and developed for 2 min (tracks 1 and 2) and a cross-reactive insert (200 ng at 3 x 108 dpm/ng), exposed for 3 days and developed for 2 min (tracks 3 and 4). Three ng of T. saginata DNA could be easily detected by both probes and 25 ng of T. solium DNA by the cross-reactive probe. Human, bovine, T. taeniaeformis, T. crassiceps and Echinococcus granulosus DNA's gave a negative result as in track 1.

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FIG. 3

Restriction map of recombinant plasmid pKYT225 which contains an insert of DNA corresponding to

the entire mitochondrial genome of Taenia hydatigena [After Yap et al. (51)]

Heavy line corresponds to vector pBR322 and light line to mtDNA of the parasite.

ECHINOCOCCUS SPP.

A number of intra-specific variants or strains of Echinococcus have been described from different geographic areas or intermediate host species (23, 46, 47). The nature and possible significance of genetic variation within the hydatid organisms have been discussed (46).

E. GRANULOSUS

Several biologically distinct strains of E. granulosus have been shown to exist which may vary in their infectivity to animals and man, with important

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implications for the epidemiology of hydatid disease. The unambiguous identification and characterisation of such strains has proved a major challenge in diagnostic parasitology. Traditionally, morphological characters have been used to discriminate strains but, in some cases, criteria such as development in vivo and in vitro, serotype, chemical composition, metabolism and enzyme electrophoresis have also been applied (20, 47). Restriction endonuclease analysis and DNA hybridisation measure genetic variation on a finer scale than previous methods and the application of this approach has provided a rapid, reproducible method not only for discriminating E. granulosus from E. multilocularis (22, 49) but also for distinguishing intra-specific variants of both species. Moreover, the technique is sensitive enough that the DNA of a single adult of E. granulosus can be characterised (26). Cloned ribosomal DNA sequences and DNA sequences selected from the E. granulosus genomic DNA library, referred to above, have proved invaluable as DNA probes in E. granulosus strain characterisation. Isolates from the United Kingdom, Africa (especially the Turkana region of Kenya), India, Western and Eastern Europe, the Mediterranean littoral, the Middle East and South America have been successfully characterised by this technique and some distinct strains identified (10, 22, 24, 25, 26, 34).

Distinguishing the "horse" and "sheep" strains

In the United Kingdom, distinct horse/dog and sheep/dog strains of E. granulosus are recognised and these differ substantially in their biological and biochemical characteristics (see 27). It is generally accepted that the latter strain is infective to man, whereas, for reasons which remain unclear, the former is probably non-infective or poorly infective to man (23). An extensive study has recently been carried out where protoscoleces from 60 individual cysts ( = isolates) of sheep origin and 64 individual cysts of horse origin were subjected to DNA analysis (26). This involved extraction of DNA from each isolate, restriction endonuclease digestion of all samples with EcoR1 and BamH1 and of 38 (19 sheep and 19 horse isolates) samples with Hind111, and Southern hybridisation with pSM889 (a cloned rDNA sequence) and pEG18. For each restriction enzyme and probe, a constant and characteristic hybridisation profile was produced with DNA from all the sheep isolates, regardless of the tissue location of the hydatid cysts. The only differences observed were some changes in relative intensity of some minor fragments. Likewise, the horse isolates all produced common banding patterns but these were quite distinct from those obtained with the sheep material. Some of the results obtained in this study are presented in Fig. 4. The analysis confirmed the earlier studies (reviewed in 27) on the morphology, developmental characteristics, chemical composition and metabolism, isoenzymes and DNA restriction fragment banding patterns of E. granulosus, which showed the distinctiveness between, but uniformity within, the sheep/dog and horse/dog strains in the UK. DNA studies along similar lines (26) suggest that the UK sheep/dog strain is cosmopolitan in its distribution and that it is genetically homogeneous; in addition, the UK horse/dog strain is genetically similar to that infecting equines in Spain and Ireland.

DNA characterisation of other strains

In common with many other parts of the world, UK bovine hydatid cysts are generally sterile or degenerate and thus are not amenable to the type of DNA analysis described above. However, a number of fertile cysts were taken from six animals originating from the Isle of Skye, North-West Scotland and the subsequent DNA

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S H C S H C S H C S H C S H C S H C kb

EcoRI BamHI

E. granulosus DNA + 3 2 P p E G 18

DNA ANALYSIS - U.K. ISOLATES

EcoRI BamHI

E. granulosus DNA + 3 2 P- rDNA

DNA ANALYSIS -U.K. ISOLATES

S, sheep origin; H, horse origin; C, cattle origin

F I G . 4

The hybridisation of 32P-labelled pEG18 (upper panel) and 32P-labeIled pSM889 (lower panel)

to Echinococcus granulosus DNA restriction fragments [After McManus and Rishi (26)]

The patterns of hybridisation clearly distinguish the horse and sheep strains and indicate a very close affinity between E. granulosus from cattle and sheep.

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analysis indicated that they conformed very closely to the sheep/dog strain (26; Fig. 4). Whether infertile cysts from UK cattle represent a different form of E. granulosus remains to be established. That cattle can harbour more than one strain of E. granulosus has been strikingly shown by DNA analysis (26). DNA hybridisation profiles for two Dutch isolates were markedly different from those obtained with fertile bovine material collected from other areas (UK, Kenya, Spain and India), which was genetically very similar to the sheep/dog strain. The developmental, morphological and biochemical characteristics of the hydatid organism from Swiss cattle are quite distinct from those described for E. granulosus of other domestic animal origin from other European countries and Australia (43). The form of E. granulosus described from cattle in Switzerland does not appear to be confined to that country (46) and it is possible that the parasite found in Dutch cattle is the same strain although comparative studies at the DNA level are required to confirm this.

Isoenzyme analysis (29) has shown that goats in Kenya harbour at least two genetic variants; the majority of goats were shown to be infected with the domestic sheep/dog strain (common variety) and this has been confirmed by DNA analysis (26). One goat isolate from the Turkana region was, however, genetically very similar to those from camels and it is probable that this represents the "rare" goat variety described earlier (29). It is likely, therefore, that in Kenya and possibly elsewhere, goats can harbour both the sheep/dog strain and the strain which infects camels.

DNA analysis (24, 26) has emphasised the distinctiveness of the strain of E. granulosus infecting camels in Kenya as was shown earlier by biochemical and isoenzyme analysis (20, 29). A similar strain appears also to infect camels in the Sudan and Somalia as DNA hybridisation profiles for the isolates from these areas were common (26). Whether man can be infected with a range of different strains of E. granulosus is open to question and there has been much interest in the camel as an intermediate host of E. granulosus, particularly in regard to its possible role as a reservoir of infection in humans (see for discussion, 24). Most experimental evidence to date (4, 29), supported by DNA data (26), indicates that the most common form infecting man is the sheep/dog strain although it has been demonstrated that humans are also susceptible to sylvatic strains of E. granulosus (13, 46). It is clear from DNA studies (24, 26) and other experimental and epidemiological evidence (29, 30), that the sheep/dog strain and not the camel/dog strain is the major source of human infection in the Turkana region of Kenya and adjacent areas. All the human isolates that have been examined, to date, in this region, both by isoenzyme (29) and by DNA hybridisation analysis (24, 26), conform to the sheep strain. Furthermore, an isoenzyme study (29) suggested that the majority of dogs naturally infected with E. granulosus in northern Turkana were infected with the sheep strain. This has been confirmed by DNA hybridisation analysis because individual adults and pooled worms taken from three different dogs in this area were of sheep strain origin (26).

Cross-infection experiments and differences in morphology, antigens and geographical distribution have suggested that a pig strain of E. granulosus occurs in eastern Europe and the Soviet Union which is distinct from that present in sheep (13). DNA hybridisation results have shown clearly that there is a common genetic variant of the parasite in pigs from Yugoslavia and Poland which appears to be quite distinct from that infecting man in Europe and elsewhere (26). This fits in well with available epidemiological evidence in relation to human disease as the pig strain, like the one from horses, is considered to have a low or no infectivity for man (31, 46). There is close DNA homology between E. granulosus from Dutch cattle and pigs

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and especially between pigs and camels (26). Whether these animals harbour the same strain is an open question; further studies, involving additional DNA probes, restriction enzymes and especially DNA sequence information, will be required to substantiate this.

On the mainland of Australia, two distinct life-cycle patterns appear to maintain E. granulosus (42). One (the domestic strain) involves sheep and farm dogs, with cattle and pigs as potential accidental intermediate hosts. The other (the sylvatic strain) involves macropod marsupials, mainly wallabies, and dingoes (44). Knowledge of how these cycles interact is critical for the implementation of control programmes (46). There is evidence from morphological, biochemical and developmental studies that the two life-cycle patterns reflect the existence of two strains on the Australian mainland (46). DNA analysis has now been performed on several isolates of E. granulosus of wallaby origin; the DNA was extracted from protoscoleces isolated from individual cysts, digested with Pst1, BamH1 and Hind111, the DNA fragments blotted onto nylon and sequentially probed with pSM889 and pEG18. Some minor differences were noted in the DNA hybridisation profiles of each isolate but the major bands were common (Fig. 5). In addition, DNA of sheep strain origin produced very similar hybridisation patterns (Fig. 5). The previously proposed existence of two distinct strains of E. granulosus on the Australian mainland may, therefore, require re-evaluation.

E. MULTILOCULARIS

In contrast to E. granulosus, there is very limited information as to possible genetic variability within E. multilocularis although isolates originating from Alaska, Europe and Siberia may show differences in morphology, pathogenicity, developmental characteristics in definitive and intermediate hosts and in their host specificity (reviewed in 13, 46). Genomic DNA's of ten E. multilocularis isolates originating from different geographical areas have been analysed by Southern blotting using the Echinococcus-speciñc pAL1, described above, as a probe (49). Restriction fragment length polymorphisms (RFLP's) were demonstrated showing, for the first time, genetic heterogeneity within E. multilocularis. Whether this DNA variation reflects differences in susceptibility to drug treatment, infectivity for different definitive and intermediate host species, differences in the biological development in infected intermediate hosts and the possible existence of discrete strains requires further study.

TAENIA SPP.

Although intra-specific variation is clearly widespread in Echinococcus, especially E. granulosus, little is known of the extent of heterogeneity in other taeniids. Such studies of Taenia spp. affecting domestic animals and man are obviously essential if an accurate assessment of their epidemiology is to be made. There is evidence that subspecific variation occurs in other taeniids (1, 33, 45) although published studies showing intra-specific DNA differences are limited to T. solium (28, 36). Thus, RFLP's were observed when T. solium genomic DNA's of Indian, Mexican and Zimbabwean

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origin were used in Southern blotting experiments with recombinant plasmids containing rDNA and T. solium DNA sequences as probes (Fig. 6). Studies along similar lines with other Taenia spp. are clearly warranted.

F I G . 6

Autoradiographs of Southern blots of Pst1- and Kpn1-derived restriction fragments

of Taenia solium DNA's hybridised with radiolabeled pTS17 (panel a), pSM889 (panel b)

[After Rishi and McManus (36)]

Each lane contained approximately 1 ug DNA. Lanes 1,4, T. solium (Indian origin); Lanes 2,5, T. solium (Zimbabwean origin); Lanes 3, 6, T. solium (Mexican origin). RFLP's are clearly evident, providing evidence for intra-specific variation in T. solium.

EGG DETECTION

The capability of distinguishing the morphologically identical eggs of the different taeniids represents an important goal in parasite diagnosis but it has proved a major technical challenge. A significant breakthrough was achieved with the development of an anti-oncospheral monoclonal antibody for the unequivocal identification of

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eggs of Echinococcus spp. by immunofluorescence (8, 9). This technique is not available, as yet, for specific identification of other taeniid eggs. Another approach, which has some advantages over an immunological assay, is a DNA-DNA dot-blot hybridisation procedure. The species-specific taeniid DNA sequences described above either provide insufficient sensitivity or have not been applied in such an assay. Two groups have, as an alternative, investigated the possibility of using total genomic DNA as a probe for distinguishing taeniid eggs. Flisser et al. (14) showed that T. saginata eggs could be detected with high sensitivity using this approach (Fig. 7). No hybridisation of T. saginata DNA with T. pisiformis eggs and vice versa was obtained, while minimal hybridisation of T. solium DNA with T. saginata DNA was observed. Experiments along similar lines by Yap et al. (51) clearly distinguished between the eggs of T. hydatigena and T. taeniaeformis. It should be noted that these tests have yet to be evaluated under field conditions for specific egg detection in faecal samples.

Procedures involving non-radioactive probes are now being developed for the identification of parasites (5) as these are more appropriate than radioactive assays for field use. One of the most widely used systems makes use of biotin, an enzyme co-factor, which can be attached to deoxynucleotides and incorporated into DNA probes. Photoactivated analogues of biotin are also available which can improve sensitivity. Nevertheless, it is clear that the non-radioactive detection assays currently available provide less sensitivity than their radioactive counterparts. A single T. saginata egg can be detected with 3 2P-labelled total genomic DNA (Fig. 7, upper panel), whereas the limit of sensitivity is 50-100 eggs using biotinylated total DNA (Fig. 7, lower panel) (14).

c D N A S E Q U E N C E S A N D B I O L O G I C A L F U N C T I O N

cDNA's encoding several taeniid cestode antigens have now been cloned and sequenced. These include a gene of T. ovis, which encodes a protective antigen and may provide the first successful recombinant vaccine against a parasite (17) and a gene encoding an E. multilocularis-specific molecule (Hemmings and McManus, unpublished). Comparison of the inferred amino acid sequence from the corresponding cDNA's of two E. granulosus antigens with other known sequences suggest that they may play an important biological role. One has a high degree of homology with the human cyclosporin A binding molecule cyclophilin (19). This may reflect an in vivo immunosuppressive mechanism ensuring, in part, the long-term survival of the parasite in infected hosts as it is known that the binding of cyclosporin A to cyclophilin in man suppresses T cell function.

Sequence is also available (Shepherd, Aitkin and McManus, unpublished) for a cDNA encoding the carboxyl-terminal of one of the major hydatid cyst fluid antigens produced by E. granulosus, the 12kDa antigen a subunit of antigen B (40). In addition, amino acid sequence has been generated for the amino-terminal which is tentatively contiguous with the open reading frame of the DNA-derived sequence (Fig. 8). The inferred sequence of the 12kDa antigen indicates a limited similarity to baboon and human alpha-1 antitrypsin. In functional assays, electrophoretically purified native 12kDa antigen from natural infections inhibited elastase but not trypsin or chymotrypsin, providing further evidence that this antigen is a parasite protease inhibitor. Possibly unrelated to its anti-protease activity but a potentially important

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F I G . 7 Specific detection of Taenia saginata eggs by DNA hybridisation

[After Flisser et al. (14)]

T. saginata and T. pisiformis eggs were obtained by mincing gravid proglotids expelled from one man and one dog respectively. Eggs were decanted and spun for 20 min at 2,000 rpm. Different concentrations of eggs in 200 ul water aliquots were prepared as follows: 5000 (A), 1000 (B), 500 (C), 100 (D), 50 (E), 10 (F), 5 (G) and 1 (H). All aliquots were boiled at 90°C for 5 min and chilled on ice. T. saginata (1) or T. pisiformis (2) eggs were dotted onto nitrocellulose membranes (NCMS) using a Bio-Rad dot blotter. NCMS were soaked in denaturing solution (1.5M NaC1; 0.5M NaOH) and then in neutralising buffer (1.5M NaC1; 0.5M Tris-HC1, pH2; 1mM Na 2EDTA) and incubated with 100 ug/ml proteinase K in 0.1 M Tris-HC1 pH 7.4 for 20 min at 37°C. NCMS were then baked at 80°C for 2 hours and incubated with prehybridisation solution (6XSSC; 5XDenhardt's solution; 0.5% SDS). Total genomic DNA (3), purified from Taenia proglotids by proteinase K digestion and phenol/chloroform extraction, was also dotted: 100 ng, 10 ng or 1 ng of T. saginata (A, B, & C respectively), T. pisiformis (D, E, & F respectively) or T. solium (G & H). Radioactive and biotinylated probes were prepared by nick translation using the manufacturer's instructions with 50 uCi of 3 2P-CTP (Amersham) or 0.1 M of biotin-labelled ATP (Bio-Rad) and 1 ug of T. saginata (I), T. pisiformis (II) or T. solium (III) DNA. Hybridisation was performed overnight at 65°C. NCMS were washed consecutively at 65°C in SSC, 0.1% SDS as follows: 2X (10 min),

(10 min), 1X (15 min), 0.1X (10 min) and 0.01X (10 min) and either exposed for 48 hours to X-ray film (upper panel) or subjected to colour reaction using the streptavidin-peroxidase (BRL) system and developing the colour for 6 hours (lower panel).

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F I G . 8

The amino-acid and cDNA sequences of the 12kDa antigen of Echinococcus granulosus present in hydatid cyst fluid

Identities between residues ascribed by amino-acid sequencing and cDNA sequencing are indicated by black crosses. X refers to the first residue that could not be identified owing to salt contamination. The bold numbers refer to number of amino acids from the amino terminus. The DNA sequence consists of 282 bases. The polyadenylation signal is underlined. Comparison of the inferred sequence of the 12kDa antigen with other known sequences indicates a limited similarity to &-1 antitrypsin. Note the mismatch between the DNA-derived sequence and the protein sequence.

function of the 12kDa antigen was its ability to inhibit recruitment of neutrophils in an in vitro assay. These functions may be important to the viability of the parasite in the face of the host immune response. In addition, the match between the DNA-derived sequence and the protein sequence is imperfect, with some residues having, according to the amino acid sequencing, two alternatives in approximately equal concentrations, and four DNA-derived residues failing to match with the protein sequence at all (Fig. 8). The 12kDa may be expressed as isoforms from a polymorphic

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gene. The detected variation in the amino acid sequence may have been exacerbated by pooling cyst fluid from several different cysts for antigen purification, thus introducing heterogeneity into the sample. This sequence polymorphism has not been reported, to date, for any other helminth antigen. It may represent an important immune evasion strategy evolved by E. granulosus and may, in part, explain why a high percentage of patients with proven clinical hydatid disease do not appear to produce antibodies to the 12kDa antigen (40).

FUTURE PROSPECTS: THE PCR REVOLUTION

There is no doubt that the polymerase chain reaction (38) has already made a considerable impact on medical and veterinary research. The research and diagnostic applications of the technique when combined with hybridisation, molecular cloning and DNA sequencing are limitless for the study of parasites. Some of the various applications of PCR in veterinary research and diagnosis have been described (11). Some uses of the technique in parasite diagnosis have also been discussed (12).

The starting point for PCR, "the target sequence", is a gene or other DNA segment. In a matter of hours this particular sequence can be reproduced (amplified) a million-fold. The complementary strands of a double-stranded molecule of DNA are separated by heating. Two small pieces of synthetic DNA (15-30 nucleotides long), each complementing a specific sequence at opposite ends of the target sequence, serve as primers. Each primer binds to its complementary sequence. A DNA polymerase (the stable Taq polymerase which comes from bacteria that Uve in hot springs, is now the enzyme of choice) starts at each primer and copies the sequence of that strand. Within a short time, exact replicas of the target sequence have been produced. In subsequent cycles, double-stranded molecules of both the original DNA and the copies are separated; primers bind again to the complementary sequences and the polymerase replicates them. At the end of many cycles, the pool is greatly enriched in small pieces of DNA that have the target sequences, and this amplified genetic information is then available for further analysis.

One of the areas where PCR will be especially useful in relation to the taeniids is the development of exquisitely sensitive diagnostic assays. For example, primers can be designed for PCR amplification of species-specific or even strain-specific DNA segments which can then be used as DNA probes for identification purposes. PCR can also be used to amplify trace amounts of genetic material directly. One application would be to strain type hydatid cysts in man or animals where few or no protoscoleces are present. It should be feasible to amplify strain-specific DNA sequences present in the germinal layer or even hydatid cyst fluid and to identify the strain by direct visualisation of the amplified product in an ethidium bromide stained gel, without subsequent blotting, hybridisation, or washing and exposure. An extension of this would be the specific detection of adult worms of Echinococcus in dogs or T. solium/ T. saginata in humans using PCR to prime species-specific sequences in eggs or tegumental material shed by the worms and present in faeces. Improved methods for extracting DNA from stool samples (7) and the development of non-radioactive PCR-based color complementation assays (6) will facilitate the use of such tests in the field.

Another area where PCR will prove especially valuable is the examination of variation within and between taeniid species in specific segments of DNA. This will

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provide a unique opportunity for phylogenetic and population research studies on the group. Conventionally, geneticists and taxonomists have used restriction endonucleases and the construction and screening of clone libraries to study molecular evolution in a variety of bacterial, plant and animal species. This approach is tedious and a fast alternative has emerged in the form of PCR, whereby unique sequences can be cloned in vitro in several hours. In combination with direct sequencing of the PCR product (15), the procedure can be automated and particular DNA sequences from many individual parasites can be routinely analysed. Potentially, many different genes or DNA segments can be compared once suitable primers have been prepared; the DNA sequence of interest may be obtained randomly from a genomic DNA library (see, for example, 48) or by selection of an appropriate DNA sequence comprising highly conserved regions separated by a semiconserved or highly variable region (see, for example, 18). An example of the latter approach is shown in Fig. 9 where, based on published sequence information (32), primers have been prepared to conserved portions of the 5' terminal domain of large subunit rDNA. The intervening DNA

1 2 3 4 5 6 7 8

FIG. 9

Photograph of an agarose gel of a PCR performed on DNA extracted from seven different isolates of Echinococcus granulosus (lanes 2-8, parasite DNA's

of sheep, horse, goat, cattle, camel, pig and wallaby origin respectively)

The amplified DNA sequence, which is arrowed, is a portion of the 5' domain of large subunit rDNA and is approximately 300 base pairs in size. Less than 100 ng template DNA was used in each lane. Lane 1 contains a BRL 1 kilobase standard ladder.

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sequence has been PCR-amplified using total genomic DNA's from different isolates of E. granulosus, and the amplified products (approximately 300 base pairs) are shown in an ethidium bromide stained agarose gel. Similarly, this sequence has been amplified from DNA's isolated from a range of taeniid species. Direct sequencing of this and other sequences will allow much more systematic determination of inter- and intra-species relationships within the group than has been possible hitherto.

ACKNOWLEDGMENTS

I am grateful to Ms Josephine Bowles for excellent technical help and Drs R.C.A. Thompson, J. Eckert and B. Gottstein for allowing me to quote unpublished information.

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CARACTÉRISATION DES TAENIDÉS PAR ANALYSE DE L'ADN. - D.P. McManus.

Résumé: L'auteur décrit les applications de la biologie moléculaire qui fournissent les meilleures méthodes d'identification et de caractérisation des Taenidés. L'auteur souligne l'importance de ces techniques pour une diagnose précise des différents stades du cycle évolutif des taenias, et pour une différenciation des espèces et des souches. Ces techniques permettent également de déterminer les liens phylogéniques, et d'identifier les fonctions des macromolécules-clés obtenues d'après les séquences d'acides aminés déduites des séquences d'ADNc clonées. L'auteur décrit en outre les méthodes mises en œuvre pour isoler l'ADN des taenias, pour cloner l'ADN génomique et mitochondrial de ces organismes, ainsi que les méthodes biotechnologiques utilisées pour caractériser les souches et détecter les œufs. Enfin, il souligne l'intérêt présenté par la réaction d'amplification enzymatique qui constitue un élément essentiel des nouvelles méthodes, extrêmement sensibles, de diagnose des diverses espèces de taenias. Associée à des techniques de séquençage direct, la réaction d'amplification enzymatique est une méthode nouvelle et efficace, qui permet d'étudier les variations génétiques entre différentes espèces de taenias, et au sein de chacune d'entre elles.

MOTS-CLÉS : Caractérisation des souches - Cestodes - Clonage de l'ADN des Taenidés - Clonage de l'ADNc de Echinococcus - Echinococcus - Réaction d'amplification enzymatique par PCR - Recherche de l'ADN des œufs -Séquençage direct - Taenia - Taenidés.

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CARACTERIZACIÓN DE CESTODOS TAENIIDAE MEDIANTE EL ANÁLISIS DEL ADN. - D.P. McManus.

Resumen: El autor describe las aplicaciones de la biología molecular que proporcionan los mejores métodos de identificación y caracterización de los cestodos taeniidae. Subraya la importancia de estas técnicas para un diagnóstico preciso de las diferentes fases del ciclo evolutivo de las tenias, así como para una diferenciacion de especies y cepas. Estas técnicas permiten también determinar los vínculos filogénicos e identificar las funciones de las macromoléculas clave obtenidas a partir de las secuencias de ácidos aminados

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deducidas de las secuencias de ADNc clonados. Por otra parte, el autor describe los métodos para aislar el ADN de las tenias y para clonar el ADN genómico y mitocondrial de estos organismos, así como los métodos biotecnológicos utilizados para caracterizar las cepas y detectar los huevos. Por último, llama la atención sobre el interés que presenta la reacción de amplificación enzimática (Polymerase chain reaction, PCR), un elemento esencial de los nuevos métodos de diagnóstico de las distintas especies de tenias, que son sumamente sensibles. Asociada a técnicas de secuenciación directa, la reacción de amplificación enzimática es un método novedoso y eficaz que permite estudiar las variaciones genéticas entre especies diferentes de tenias y dentro de cada una de estas especies.

PALABRAS CLAVE: Caracterización de cepas - Cestodos taeniidae -Clonación del ADN de Taeniidae - Clonación del ADNc de Echinococcus -Detección del ADN de los huevos - Echinococcus - Reacción de amplificación enzimática - Secuenciación directa - Taeniidae - Tenia.

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