Isolation and characterisation of nucleic acids from the hydatid organisms, Echinococcus spp. (Cestoda)

Molecular and Biochemical Parasitology, 16 (1985) 251-266 Elsevier

251

MBP 00573

ISOLATION AND CHARACTERISATION OF NUCLEIC ACIDS FROM THE HYDATID ORGANISMS, ECHINOCOCCUS SPP. (CESTODA)

DONALD P. McMANUS', MATTY KNIGHT 2 and ANDREW J.G. SIMPSON 2

tDepartment of Pure and Applied Biology, Imperial College of Science and Technology, Prince Consort Road, London SW7 2BB, and 2Division of Parasitology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K. (Received 27 March 1985; accepted 23 April 1985)

DNA and RNA in combination have been prepared and characterised from the hydatid disease organisms, Echinococcus granulosus and Echinococcus muitilocularis. The DNA obtained is of high molecular weight, pure and can be cleaved by restriction enzymes, thereby facilitating future production ofgenomic DNA probes for studies of Echinococcus gene expression. Moreover, cloned DNA segments from Schistosoma mansoni hybridise strongly to Echinococcus DNA following restriction and Southern blot analysis. The extracted RNA is functional and has been translated in vitro. The major translated polypcptides and antigens have been identified, and the technique can now be used to analyse differential gene expression during development and differentiation of the hydatid organisms and to identify specific polypeptide antigens which may have potential as immunodiagnostic reagents. Key words: Echinococcus granulosus; Echinococcus multilocularis; Hydatid disease; DNA; RNA; In vitro translation

INTRODUCTION

Recent developments in recombinant DNA and bacterial cloning technology [ 1-3], open up exciting new avenues for the study of parasites, and, in particular, for investigating the complex interactions they have with their hosts. Molecular approaches are already providing powerful probes for the identification and characterisation of gene products of an increasingnumber of medically important parasites, including Plasmodium (see, for example refs. 4--6), Trypanosoma (see, for example refs. 7, 8) and Schistosoma [9-16]. To date, however, there have been no attempts to apply this technology to the genomic structure and organisation of any cestode parasite of man, or to study the molecular characteristics of the host/parasite relationship in human cestodiases. This reflects, in part, the slow progress which has been made in the field of nucleic acid biochemistry of tapeworms in general. RNAs from the metacestode (larval) stage of both the rodent cestode, Taenia crassiceps [17] and the 'pig' tapeworm, Taenia solium [18] have been extracted which 0166-6851/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

252 direct the synthesis of parasite, parasite-like or, in the latter case, host-like antigens in cell-free heterologous systems. Recently, a significant advance was made by Saint and his co-workers, who produced a cDNA expression library from mRNA isolated from the rodent parasite, Taenia taeniaeformis in the bacteriophage vector k gt 11 [19], using protocols developed for the expression of Plasmodium antigens in Escherichia coli [20]. Three immunologically-distinct T. taeniaeformis antigens expressed in E. coli were identified. The limited work carried out on nucleic acids and nucleic acid metabolism in the hydatid disease organisms, tapeworms belonging to the genus Echinococcus, has been recently reviewed by one of us [21]. In particular, RNA from Echinococcus granulosus, which causes unilocular hydatidosis in man, was comprehensively analysed in terms of nucleotide composition, sedimentation properties, template activity and biosynthetic kinetics [22] although there was no molecular characterisation of the isolated mRNA in that study. Additionally, a homologous cell-free system capable of synthesising proteins has been prepared from this species [23], but no attempt has been made to characterise the TCA-precipitable peptides formed from the in vitro translation of total RNA. As a prerequisite for applying recombinant DNA techniques to Echinococcus, we describe a method for the isolation of DNA and RNA from protoscoleces of E. granulosus and the related E. multilocularis. E. multilocularis is the causative agent of alveolar hydatid disease in man. The nucleic acids are obtained in pure form, and are functional. The DNA can be spooled, cleaved by restriction enzymes, and can serve for Southern blot analysis while the RNA can programme protein synthesis in vitro. The method is especially useful because it combines the extraction of both nucleic acid species from a small amount (mg quantities) of hydatid material. Hydatid cysts are often at a premium when collected from abattoirs, or especially at surgery, and this relative lack of parasite material has severely restricted biochemical research on the hydatid organisms in the past. MATERIALSAND METHODS

Parasites. Protoscoleces of E. granulosus (horse and sheep strains - see McManus and Macpherson [24]) and E. multilocularis were obtained from hydatid cyst material by treatment in Hanks' saline, pH 2.0, containing 0.2 or 0.5% (w/v) pepsin, followed by several rinses in Hanks' saline [25]. Worms were finally suspended in a minimum volume of Hanks' saline and stored in liquid nitrogen or at -70°C. Materials. Restriction endonucleases were purchased from either Boehringer Mannheim or Bethesda Research Laboratories. Digestions were carried out in enzyme excess with the conditions recommended by the manufacturers. [35S]Methionine, [a-32P]dCTP and rabbit reticulocyte lysate (nuclease treated) were obtained from the Radiochemical Centre, Amersham, England. All other chemicals were of analytical

253 grade, and were obtained, unless otherwise described, from the Sigma Chemical Company or BDH Chemical Company, Poole, Dorset. All glassware used in the experiments described in this communication was rendered RNAse-free by rinsing in double-distilled water and oven-heating at 160°C overnight prior to use. All stock solutions were autoclaved or Millipore filtered (Millipore Corp., Bedford, MA, 0.45 lam pore size) before use.

Preparation of nucleic acids from E. granulosus and E. multilocularis. Tota) nucleic acids were isolated following digestion of parasite material in sodium dodecyl sulphate (SDS) and proteinase K, followed by phenol extraction and ethanol precipitation. DNA was obtained by caesium chloride gradient centrifugation in the presence of ethidium bromide as previously described for Schistosoma mansoni [9]. An alternative procedure, using the differential solubility of DNA and RNA in lithium chloride [26], was exploited to isolate both nucleic acids in combination from the same starting parasite material. Ribosomal and messenger RNA were precipitated by this salt and the DNA, which remained in solution, was subsequently precipitated in ethanol. This isolation procedure is outlined in detail in Fig. 1. In vitro translation ofEchinococcus RNA. Total RNA samples (3 ~tg) were translated in vitro in a reaction volume of 18 lal in the presence of [35S]methionine for 90 min at 30°C using rabbit reticulocyte lysates as previously described by Pelham and Jackson [27]. Following each translation, incorporation of [35S]methionine into proteins was assessed by liquid scintillation counting and the radiolabelled polypeptides were directly analysed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fluorography, or were subjected to immunoprecipitation before analysis. Immunoprecipitation of [3SS]methionine-labelled polypeptides. Human infection serum was obtained from a Kenyan (Turkana region) patient, chronically infected with E. granulosus. Samples of the translation products (1-3 ~tl) were added to 5 ~tl of this antiserum in a final volume of 50 ~tl made up with 0.05% Triton X-100, 50 mM Tris-HC1, 150 mM NaCI, 10 mM EDTA, pH 8.0 (precipitation buffer). After incubation overnight at 4°C, 50 lal of 50% (v/v) protein A-Sepharose (Pharmacia) in precipitation buffer was added, and the samples incubated for 60 min at room temperature. The beads were then washed four times with ice-cold precipitation buffer by centrifugation, suspended in 25 ~1 of electrophoresis buffer (1% (w/v) SDS, 50 mM Tris-HCl, 10% (v/v) 13-mercaptoethanol, 2 mM phenylmethylsulphonyl fluoride (PMSF), 1 mM ethylenediaminetetraacetic acid (EDTA), 1% (v/v) glycerol, pH 7.5), boiled for 4 min to solubilise bound antigens and electrophoresed. Electrophoresis. Restriction fragments, resulting from digestion of Echinococcus DNA (5 I~g)with restriction endonucleases, were separated by horizontal electrophoresis (25 V constant voltage, 18 h) on 0.8% (w/v) agarose slab gels.

254 ISOLATION OF' RNA AND DNA FROM ECHINOCOCCUS

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

I Powder thawed into 4 ml extraction buffer.

I

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

I

Incubate for 3 h.

I

Phenol extraction, back extraction and re.extraction.

I

Nucleic acids in supernatant precipitated in 0.3 M sodium acetate and 2.5 vol. ethanol overnight at -20°C and pelleted at 100~ × g, 10 rain, 0°12.

I

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

I A

3 vol. of 4 M LiCI added and left overnight.

RNA precipitate pelleted at 10000 × 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. Washed in 70% ethanol, dried and dissolved in 0.5 ml TE buffer.

Fig. 1. A method for the combined isolation of RNA and DNA from protoscoleces of Echinococcus. Protoscoleces were removed from hydatid cysts and freed from brood capsules using the techniques described by McManus and Smyth [25]. Typical yields using this method were similar for E. granulosus and E. multilocularis, being approximately 1.5 mg DNA and 0.75 mg total RNA. The OD~60/OD280 ratios were approximately !.8 and > 2.0 respectively, thus indicating the virtual absence of protein and other contaminants in these preparations. Extraction buffer: 50 mM Tris-HCl, pH 8.0, containing 50 mM EDTA and 100 mM NaCI. T.E. buffer: 10 mM Tris-HCl, pH 8.0, containing 1 mM EDTA.

R N A p r e p a r a t i o n s w e r e e l e c t r o p h o r e s e d in 1.5% a g a r o s e gels u n d e r d e n a t u r i n g a n d n o n - d e n a t u r i n g c o n d i t i o n s [28]. D e n a t u r e d R N A s a m p l e s w e r e p r e p a r e d in e i t h e r 5 0 % ( v / v ) d i m e t h y l s u ! p h o x i d e in 10 m M p h o s p h a t e b u f f e r , p H 6.8 o r in 1 M g l y o x a l / 3 M u r e a in 10 m M p h o s p h a t e b u f f e r , p H 6.8 a n d w e r e h e a t e d at 5 0 ° C f o r 1 h p r i o r to

1

l

255

loading. Non-denatured samples wet~ p l ~ : ~ d i~t~t0 mM phosphate buffer, pH 6.8, without heating prior to loading. E. cMt ~ , J, ~t~nsoni RNA species were used as markers. Electrophoresis was carried oat [~i~, ~[~ ~hosphate buffer, p H 6.8 at room temperature at 80 v for 4 h, taking ~ird [b ~lt't~Llldte the tank buffer to prevent the production of a pH gradient in the i~eis, ~11: i The DNA restriction fragments add t l ~ , ~lLt~lltions were stained with ethidium bromide (1 ~tg ml-~) and were photogra~tt~ d~idlJi"Uv illumination (302 nm) using an orange filter. ~[i Translation products and immunopreclblt~t~i lt~i'e electrophoresed through 12.5% (w/v) polyacrylamide slab gels [291 ariO ~Jd~b|httihed [30].

Filter hybridisation. DNA restrictidn frlt~i(liil *ere transferred to nitrocellulose filters according to the method of Sttul~hJt'laI~t], ~tilters were hybridised in 30 ml of 0.4% polyvinylpyrrolidone, 0.4% F!i:&li fl.~]J~~tiVine serum albumin, 4 X 0.15 M NaCI/0.015 M sodium citrate (SSC), ilncl~ ! ~ 1 ~ , A cocktail of denatured 32P-labelled cloned DNA segments of the ribo]om~[| ~ 1 ~ tltltie of S. mansoni [1 I] (approx. 1 ~tg, 1 × 107 cpm, labelled with [a-~XP]dCTP ~¢ tt{~ [~'anslation [321) was added in the presence of 100 ~tgdenatured, sheared c ~ t i f ' i ~ a [ ~ i A . Hybridlsation was carried out at 65°C overnight after which the filtdrs .Wli~JWtied for 2 h in 0. i X SSC, 0.05% SDS at 52°C with at least three changes. ~tae ftt[~tl ~ r e finally dried and exposed for autoradiography using Kodak X-ray dim sacked Cronex lightening plus intensifying screens. RESULTS

DNA preparation by centrifugation th~o~ ~1~tle~l~M ehloride/ethldium bromide gradient. The banding of DNA followidg gd~lt~lt~ttdti'of total nucleic acids for 18 h at 45 000 rpm in a Beckman VT1 50 rotdt" il ~hgwri i~ FIIj, 2. Two distinct bands of DNA were visualised by UV illumination, slid i~heil~ 0C~{~ed at the same regions of the gradient with preparations from E. mtfftitt~M~ls iAlid(tom both strains of E. granulosus. The DNA in the upper band was Sl~Ot~lab~,t'll~e~t~contaminating u v absorbing material ( O D 2 6 0 / O D 2 s 0 ----- 1.8) and the ~,i~la 0~ D~A prepared in this way was approximately 1 mg ml-~ packed prdt~¢bieel~s, Ill ~fltrast, the DNA in the lower band was not spoolable and was bottn~ [~ some 00tl[atrlinating polysaccharide and other unidentifiable components (see b~ltJw). ~eitrig~|on enzyme and Southern blot analysis could not distinguish betweeh iheiD~A! ttl~t~eupper and lower bands (data not shown). Consequently there was nO ~i~l~t~le thjit the DNA in the latter band represented distinct mitochondrial Di~l)k.~[l ~ther ~ i l t t e bands were observed and thus any mitochondrial DNA extracted is [~ierel'~e llitety to remain a component of the final DNA preparations.

Combined isolation ofDNA andRNA.

S~v~rttldifferent extraction procedures, includ-

256

Fig. 2. The banding of D N A from protoscoleces of Echinococcus on a caesium chloride/ethidium bromide gradient following centrifugation for 18 h at 45000 rpm in a Beckman VT1 50 rotor. The D N A was visualised by UV illumination. Note the two arrowed bands of DNA.

ing that described by Taylor et al. [14] for S. mansoni RNA, proved unsuitable for the preparation of RNA from Echinococcus. Consequently a novel technique had to be devised involving treatment of pulverised worms with Proteinase K and SDS, purification and collection of total nucleic acids, with the subsequent isolation of RNA by precipitation in LiCI (Fig. 1). The technique has the particular advantage that DNA is spoolable from the final solution and thus both nucleic acids can be obtained in tandem. Typical yields using this method were similar for E. granulosus and E. multilocularis, being approximately 1.5 mg DNA and 0.75 mg total RNA from 1 ml packed worms. The OD260/OD2s0 ratios were approximately 1.8 and > 2.0 respectively, thus indicating the virtual absence of protein and other contaminants in these preparations. The purified DNA obtained by this method appeared to band predominantly at one region of density (i.e. equivalent to the upper band) when centrifuged in a C s C I 2 gradient of the type described earlier. In this respect, a white precipitate, produced in the final solution following DNA spooling, was presumed to be the same contaminating material described above in the alternative method for DNA isolation. Analysis with the anthrone reagent [33] indicated that about 15% of this precipitate

257 was composed of polysaccharides, but the remaining components were not identified. The protocol has been shown subsequently to be appropriate for the combined isolation of D N A and R N A f r o m a range of other cestodes including T. crassiceps, Hymenolepis diminuta and Mesocestoides corti (A.K. Rishi and D.P. McManus, unpublished results). Moreover, the method may have universal application in that intact and functional nucleic acids have also been extracted f r o m mouse liver (A.K. Rishi, pers. comm.) and the nematode, Trichinella spiralis (A. Chambers, pers. comm.).

Restriction endonuclease digestion o f Echinococcus DNA. The Echinococcus D N A produced by both the described isolation methods could be restricted by a range of restriction enzymes, including Hind III, Pst 1, Eco R1 and Bam H1. Electrophoretic separation of the resulting restriction fragments following complete digestion by these enzymes produced complex patterns composed of a large number of distinct bands within the smear of D N A (Fig. 3). There were detectable differences in these patterns,

4567

i

Fig. 3. Electrophoretic separation of DNA fragments derivedfrom protoscolecesof Echinococcuson a 0.8% agarose gel following complete restriction with Hind III (lanes 1-3) and Pst 1 (lanes 4-6). Each lane contained 5 p.gof DNA. The DNA was visualisedby staining with ethidium bromide and UV illumination. Lanes 1, 4, DNA fragments from E. granulosus(sheep strain); lanes 2, 5, DNA fragmentsfrom E. granulosus (horse strain); lanes 3, 6, DNA fragments from E. multilocularis; lane 7, molecular weight markers (kilobases).

258

not only between the DNA frot B $h¢ SwplI~cies of Echinococcus,but also between the DNA of the horse and sheep st~'a~il!of~., ~at~ulosus, which could serve to distinguish these organisms. However, a m0~ i~n~i!¥ ~ ~neaos of distinguishing these parasites, by genetic analysis, requires analyl~s Of !~gtij~dual polymorphic genes. In order to carry out such an analysis, it is nectsnpd }~ $~4~,$[erthe restricted DNA onto nitrocellulose filters and to probe with a clc)laCdSOStt~l~[~f radio-labelled DNA. Experiments along these lines were carried out tO ¢)nltU~~$t]}~e isolatedEchinococcusDNA was suitable for this analysis, and to investi$~l)},Wh~la~ a 3:P-labelled cocktail of cloned segments of the ribosomal RNA gene of *, " l ~ i ~ i ~ 11] Could be used as a probe (Fig. 4). The a

b

D

uno-

1

2

3

.4.5

BamHI E,co,RI Fig. 4. Electrophoretie separation of BN~ ~Bsments derived from protoscoleces of E. mulalocularis following complete restriction with ~ $ ~ l ~ Eco RI (panel a). Each lane contained 5 ~g of DNA. The DNA was visualised by staining ~it~ ! . t ~ [ g ~ hromide and UV illumination. Autoradiograph of a Southern blot of these fragments h ~ ) ' ~ ' i ~ a cocktail of "P-labelled cloned DNA segments of the ribosomal RNA gem of S. mansoni{i[ i' 0 ~ i ~)' Lane 1, Molecular weight markers (kilobases); lanes 2 and 4, DNA isolated by centrifugation t ~ r ~ ~ $ u m chloride; lanes 3 and 5, DNA isolated following LiCI treatment of total nucleic acids. ~--

259 isolated and restricted Echinococcus D N A exhibited a highly complex pattern o f hybridisation with the S. mansoni p r o b e suggesting an extensive sequence h o m o l o g y between the ribosomal genes o f the two organisms, and indicating that this very sensitive a p p r o a c h m a y be a p p r o p r i a t e for the detection o f inter- and intraspecific D N A differences in Echinococcus. Characterization ofE. granulosus RNA. Electrophoretic analysis o f the R N A extracted f r o m the horse strain o f E. granulosus is s h o w n in Fig. 5. The gel shows no sign o f R N A degradation indicating that the isolation m e t h o d e m p l o y e d effectively inhibits e n d o g e n o u s ribonuclease activity. The R N A subjected to n o n - d e n a t u r i n g conditions (10 m M p h o s p h a t e buffer, p H 6.8) was resolved into two ribosomal species with a p p r o x i m a t e molecular weights o f 800 000 and 1600 000. W h e n the R N A was dena-

Fig. 5. Agarose-gel electrophoresis of E. granulosus(horse strain) RNA. The samples in lanes 1,2, 7, 16have been prepared in 10 mM phosphate buffer, pH 6.8 and are not denatured. The samples in lanes 3, 4, 8, 13-15 have been denatured in 50% dimethylsulphoxide in 10mM phosphate buffer, pH 6.8. The samples in lanes 5, 6, 9, 10-12 have been denatured in 1 M glyoxal/3 M urea in 10 mM phosphate buffer, pH 6.8. Lanes 1, 3 and 5, E. granulosustotal nucleic acids (10 lag); lanes 2, 4 and 6, E. granulosusRNA (5 ~tg); lanes 7, 8 and 9,E. coli RNA (10 I~g);lanes 10-16, S. mansoni RNA (10 l~g). The E. coli RNA is included as a molecular weight marker; the larger subunit is approximately 1000000 and the smaller subunit is 500000. The RNA was visualised by staining with ethidium bromide and UV illumination.

260 tured, the ribosomal R N A migrated as a single species, with an approximate molecular weight of 800 000. These results suggest that E. granulosus has an in vivo nick in the large ribosomal R N A sub-unit. A similar nicked RNA had been noted in a range of organisms, including S. mansoni [12], and R N A from this parasite was thus run concurrently for comparison (Fig. 5).

In vitro cell-free translation of Echinococcus RNA. Translation of the total RNA from the horse strain of E. granulosus in a heterologous rabbit reticulocyte lysate cell-free system [27] gave significant incorporation of [ ' S ] m e t h i o n i n e into synthesised proteins. The level of stimulation, with a series of different R N A preparations, ranged f r a m 5-10 fold over the level of endogenous incorporation measured when no R N A was added to the lysate (Table I). Similar levels of stimulation were obtained with total RNA preparations from the sheep strain of E. granulosus, E. multilocularis and T. crassiceps. When the in vitro translation products of the horse strain ofE. granulosus were analysed by autoradiography of SDS°PAGE gels, many polypeptides ranging in approximate molecular weights from < 10 000 to > 205 000 were observed (Fig. 6a,b). Interestingly, the majority of the most abundant polypeptides have a narrow range of molecular weight between 40000 and 116000. Immunoprecipitation of these translation products with h u m a n infection serum from a chronic hydatid case demonstrated that many of the synthesised proteins were recognised as antigens (Fig. 6a,b). A major, low molecular weight antigen (14 000) m a y be of potential use in immunodiagnosis of hydatid disease, as pooled h u m a n infection serum from a group of Kenyan patients chronically infected with S. mansoni failed to immunoprecipitate this antigen (Fig. 6b). Moreover, the h u m a n hydatid antiserum recognised the antigen amongst cell-free

TABLE I The incorporation of [3~S]methionineinto protein in a rabbit reticuloeyte lysate systemas directed by eight different RNA preparations from E. granulosus(horse strain) RNA preparation

[35S]methionineincorporation (cpm) pl-~

1

48

2 3 4 5 6 7 8 No RNA

145 75215 65 185 44035 36190 56 830 68805 71890 7480

3 pg of total RNA was added in each case and translation mixtures(18 pl) were incubated for 90 min at 30°C.

261

a

1234

5 6 7 8 9 10 11 12 13 14

142

3

456

Fig. 6. In vitro translation of RNA from E. granulosus. (a) [3sS]Methionine-labelled RNA translation products from three different RNA preparations from E. granulosus of horse strain origin were analysed by SDS-PAGE before (lanes 1-3) and after immunoprecipitation with human hydatid antiserum (lanes 5--7). Immunoprecipitation of translation products of two additional RNA preparations are also shown (lanes 8, 9). Note that the total translation products appear to be identical in each case as do the subsequent immunoprecipitation products. Normal human serum (lane 10) and no message blank (lane 4) were run concurrently. In addition, translation products (lanes 13, 14) and immunoprecipitation products with the human anti-serum (lanes 11, 12) directed by RNA from E. multilocularis and T. crassiceps, respectively, were run for comparison. The distortion in lanes 5-12 of approximately 50000 molecular weight is due to the immunoglobulinheavy chain used for immunoprecipitation. (b) RNA translation products of an additional RNA preparation from E. granulosus of horse strain origin was analysed by SDS-PAGE before (lanes 2, 3) and after immunoprecipitation with human hydatid antiserum (lane 6) and human S. mansoni antiserum (lane 4). Note the Echinococcus-specific low molecular weight antigen (14000) recognised only by the hydatid antiserum. Normal human serum (lane 5) and no message blank (lane 1) were run concurrently. t r a n s l a t i o n p r o d u c t s d i r e c t e d b y R N A f r o m E. multilocularis b u t a p p a r e n t l y n o t b y R N A f r o m the closely r e l a t e d t a e n i i d cestode, T. crassiceps (Fig. 6a). DISCUSSION It is clear t h a t the i m p l e m e n t a t i o n o f r e c o m b i n a n t D N A a n d r e l a t e d t e c h n i q u e s i n m o l e c u l a r b i o l o g y will g r e a t l y i m p r o v e o u r k n o w l e d g e o f the b i o l o g y o f Echinococcus

262 and hydatid disease. The protocol devised for the combined extraction of restrictable DNA and intact, functional RNA, coupled with characterisation studies of the type described in this communication are prerequisites for such future developments. The immunodiagnosis of hydatid disease suffers greatly from problems of supply of parasite material and purity of antigen preparations. In addition, these antigens are extremely cross-reactive, especially with those of other taeniid cestodes [34]; this is an acute problem in serodiagnostic tests with domestic animals. Cloning of genes encoding proteins such as'antigen 5' [35] which has the highest known degree of specificity, or other as yet uncharacterised antigens, with their subsequent expression in micro-organisms, may provide abundant antigenic proteins for immunodiagnostic tests, or for use as experimental vaccines. As the basis of a new approach for producing additional Echinococcus-specific protein antigens, we have translated the RNA from both E. granulosus and E. multilocularis and identified the major polypeptides and antigens. Encouragingly we have found a major low molecular weight antigen, which may be genus-specific and, therefore, of potential use as an immunodiagnostic reagent. Genetic engineering may well provide an even more sensitive means of diagnosing hydatid disease in that cloned DNA fragments of Echinococcus may be used directly to detect parasite DNA in hydatid-infected hosts. DNA occurs in hydatid cyst fluid [36], although whether it originates from the parasite, host or both has not yet been established. However, the germinal membrane, from which the brood capsules and protoscoleces develop by asexual budding, is, in addition to the protoscoleces, rich in DNA [37]. It is therefore likely that at least some of the cyst fluid DNA is of parasite origin. Moreover, there is a bidirectional passage of various components, including host immunoglobulins [38,39] and parasite antigens [40], between hydatid cyst fluid and host serum. Consequently, if Echinococcus DNA can similarly permeate the various layers which compose the hydatid cyst, it should be feasible to detect it subsequently in host fluids by genetic techniques. A particular problem of unilocular hydatidosis, only recently recognised, is that E. granulosus exists as different strains in different vertebrate hosts [24]. A similar strain complex may also occur within E. muhilocularis, although the evidence for the existence of such strains is not as clearly defined. Strains of E. granulosus are known to differ in their infectivity and pathology to various hosts including man and in their antigenic characteristics and susceptibility to chemotherapy [24]. The highly complex picture of strains emerging from areas such as Kenya requires that more specific and sensitive methods be employed in future than those currently in use. It is clear from genetic characterisation studies mainly with parasitic protozoa [41,42] that cloned DNA probes can provide the requisite precision reagents. In particular, the construction of a DNA library and isolation of strain-specific sequences offers an unrivalled rapid and precise method for strain characterisation ofE. granulosus (and E. multilocularis). The development of species-specific molecular probes also opens up the possibility of producing either a radioactive, enzymic or fluorescent assay for nitrocellulose filter (dot blot) [43] or microscope slide hybridisation identification of Echinococcus

263 (and other taeniid cestode) eggs. This has long been regarded as one of the major problems in diagnostic parasitology [44]. As a basis for future development of specific Echinococcus recombinant DNA probes we have shown that cloned DNA segments from S. mansoni hybridise strongly to Echinococcus DNA during restriction and Southern blot analysis. In a future publication (D.P. McManus and A.J.G. Simpson, in prep.) we will show that significant inter- and intraspecific differences are apparent in Echinococcus using this sensitive approach, whereby these organisms can be identified. At the present time, surgery remains the treatment of choice for human hydatidosis, and this can result in a poor prognosis. Moreover, alveolar hydatid disease is often not diagnosed until the disease is well advanced, and the parasite lesion is often inoperable. Although there are signs of promise for chemotherapy of unilocular hydatidosis [45], there is still no practical chemotherapeutic treatment available. Future application of molecular technology may improve prospects for the developments of more effective drugs for treatment of hydatid disease in man. For example, gene cloning may provide information on sequence and structural properties of key proteins involved in growth, development, differentiation, metabolism or transport of nutrients in Echinococcus which could lead to chemotherapeutic targeting. An alternative strategy for detecting proteins in Echinococcus susceptible to chemotherapy, would be to identify genes coding for enzymic, secretory or structural polypeptides which could be shown by genetic techniques to be significantly different from the analogous host proteins. In addition to these practical considerations, Echinococcus represents an excellent model for the study of development and differentiation during a parasite lifecycle because the protoscoleces have the unusual potential of being capable of developing in one of two directions, either (a), in a cystic direction, resulting in asexual differentiation - as in the case of protoscoleces which leak from a cyst into the body cavity to form secondary hydatid cysts or (b), in a strobilar direction - as in the case of protoscoleces taken into the gut of a dog where they undergo sexual differentiation, strobilate and develop into adult worms. Moreover, the direction of development can be controlled under suitable conditions in vitro [46]. Currently, nothing is known concerning the unusual growth characteristics of Echinococcus, but as a result of the work described in this communication, we are now in a position to analyse, in detail, differential gene expression at different life cycle stages of the hydatid organisms. ACKNOWLEDGEMENTS Much of this work was performed in the Division of Parasitology, N.I.M.R., Mill Hill, London, and the authors would like to thank Miss Maliha Chaudri for her help with technical aspects of this work and Dr. S.R. Smithers for encouragement. D.P.M. acknowledges generous financial support from the Wellcome Trust and E.E.C.

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