Infernational Journal for Parasitology.
1978. Vol. 8. pp. 259-265. Pergamon Press. Printed
ECHINOCOCCUS
BETWEEN ANTIGENS SILVIO
GRANULOSUS
in Great
Britain.
: COMPARISON
IN SCOLICES AND HYDATID
FLUID
DOTTORINI and CARMELO TAM
Institute of Infectious Diseases, Perugia University, Perugia, Italy (Received 3 1 March 1977, in revised form 24 October 1977) Abstract-DOTTORINI S. and TASS C. 1978. Echinococcus granutosus: comparison between antigens in scolices and hydatid fluid. International Journal for Parasitology 8: 259-265. The sheep hydatid fluid and scolex antigens of Echinococcus granulosus were precipitated by increasing ammonium sulphate concentrations. The antigenic profiles, obtained by complement fixation and indirect hemagglutination inhibition tests on the ammonium sulphate precipitates after linear sucrose gradient ultracentrifugation, were different comparing the hydatid fluid and the scolex extracts. Antigenic nonidentity was found between sheep hydatid fluid and scolex extracts by immunodiffusion and indirect hemagglutination inhibition tests. The ammonium sulphate precipitates of hydatid fluid and scolex extracts revealed several different bands by slab-gel examination.
INDEX KEY WORDS: Echinococcus granulosus; sheep hydatid fluid; hydatid scolices; immunolelectrophoretic analysis; sucrose gradient separation; slab-gel characterization; immunodiffusion test; indirect hemagglutination, indirect hemagglutination inhibition and complement fixation reactions.
INTRODUCTION
MATERIALS
AND METHODS
Hydatid scolex antigens. The scolices from ovine hydatid cysts were diluted 1:4 with saline solution, frozen with dry ice and thawed twice, sonicated for 5 min at full power in an MSE 10 Kc Sonic Oscillator and dialyzed for 48 h, then centrifuged at 16,OUOg for 15 min. The supsrnatant was saved and the pellet was resuspended with saline solution and sonicated for 3 min, centrifuged again for 15 min at 16,000 g (final pzllet = SC,). The two supzrnatants were recombined (SC,). A part of SC, was treated with increasing ammonium sulphate concentrations and the following pellets were collected: 0.8 M (SC,), 1.6 M (SC,), 2.5 M (SC,) and 5.0 M (SCG) by centrifugation at 16,000 g for 15 min and dialysis for 16h. Hydatid fluid antigens. The same treatment of the scolex antigens extraction was p:rformed on fertile sheep hydatid fluid. The so-called ‘globulinic antigen’ of Lorenzo (1960), according to the technique of Lorenzo (1960), was obtained from hydatid fluid, through precipitation at half saturation of (NHJ2S04. Linear sucrose gradients. The antigenic sample (0.5 ml) was layered on 4.5 ml of continuous sucrose gradients formed with a mixing device from 15 to 45% (w/v) sucrose (Analar) in tris-HCI buffered saline pH 7.2 and centrifuged at 105,000 g for 17 h at 4’C on the SWSOL rotor in a SPINCO L2-50B preparative ultracentrifuge. Twelve-drop fractions were collected by the Density Gradient Fractionator ISCO. SDS Polyacrilamide gel electrophoresis on slab-gel. Antigenic samples were precipitated with 20% TCA, washed, dissolved in 5 vol. of 5% (w/v) SDS, 5% (v/v) B-mercaptoethanol, 0.05 M-tris-HC1, pH 6.8, 10% (v/v) glycerol, 0.01% (w/v) Bromophenol blue; boiled for 4 min and applied to gel composed of 15 % (w/v) acry-
SHEEP hydatid fluid is a mixture of sheep serum components and parasite antigens (Kagan & Agosin, 1968). For the serologic diagnosis of human hydatid disease, fluid collected from hydatid cysts of infected sheep is the antigen currently employed. Scolex antigens, although composed mainly of parasitic antigenic components, are not very reactive in serologic tests (Kagan, 1963).
It seems important to separate and identify the different parasite antigens of hydatid fluid and scolices in order to study the human and animal antibody response towards the most important of them. Besides, some high mol. wt. fractions, always present in scolices and seldom in hydatid fluid, are immunogenic against the secondary, experimental hydatid disease in mice (Pauluzzi, Dottorini, De Rosa & Tassi, in preparation). In this work we proposed to characterize comparatively the antigens of ovine hydatid fluid and scolices. In order to do this we examined the fractions obtained by ammonium sulphate salting out and linear sucrose gradient ultracentrifugation by various serologic methods (complement fixation, immunodiffusion, indirect hemagglutination inhibition) and slab-gel electrophoresis. Investigations were carried out to establish whether separation techniques of various antigenic fractions are reproducible and whether identity or non-identity between antigens from hydatid fluid and scolices exists. 259
260
SILVIODOTTORINIand CARMELOTASS
lamide and 0.085% (w/v) bis-acrilamide. A slab-gel apparatus (Studier, 1973) was employed for the electrophoresis. Samples were electrophoresed for 3 h at 10 V/ cm at room temperature. After electrophoresis, the gel was stained with .05 % Coomassie Brillant Blue in 10% acetic acid and 30% methanol for 30 min at 60°C and destained overnight at room temperature in the same solvent. lmmunoelectvophoresis (I.E.P.). We followed the technique of Scheidegger (1955) by the Buchler apparatus (Fort Lee, NJ, U.S.A.). Agarose (Industrie Biologique Francaise) was used at 1.1% with Verona1 buffer pH 8.6 ionic strength 0.025 M, on glass slides 75 x 25 cm. The electrophoretic migration was carried out at 6.7 V/cm for 120 min: the slides were stained with Amido Schwarz 10B. Immunodifision (I.D.). The technique of double diffusion by Ouchterlony (1958) was used. Immune seru. A pool of human sera was made (PSD,, ‘reference serum’), drawn from subjects infected with hydatidosis and stored at -20°C until used. The titer (1:819,000) of the ‘reference serum’ was determined with sheep RBC coated with the ‘globulinic antigen’ of Lorenzo (1960), according to the technique mentioned in the following paragraph. Rabbit anti-sheep serum (Cappel Laboratories, Downingtown, PA 19335, U.S.A.) was used to differentiate host proteins. Indirect hemagglutination (I.H.A.), I.H.A. inhibition, and complement fixation (C.F.) reactions. The reaction of I.H.A. and I.H.A. inhibition was used according to the technique of Czimas (1960), Boyden (1951) and Pauluzzi (1969), as previously described in a paper by Pauluzzi, De Rosa & Dottorini (1971). Sheep red blood cells, treated with formalin, were sensitized with ‘globulinic antigen’ prepared according to Lorenzo (1960) which had a protein content of 5 mg/ml; while the protein content of the scolex extracts ranged between 4.0 and 1I .O mg/ml. The range of greatest reactivity was obtained,
320 C F
I
A
I.J.P.
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1978
by checkboard titration, at an antigen concentration of 0.5 mg/ml which was used thereafter; the antiserum utilized was PSI), ‘reference serum’. For the Complement Fixation reaction, the micromethod of Sever (1962) was carried out using overnight fixation at 4°C with two exact units of complement and four antibody units. Chemical test. The protein content of antigens was determined by the method of Lowry, Rosebrough, Farr &Randall (1951). RESULTS The antigenic activity of the scolex and hydatid fluid fractions precipitated at 0.8-1.6-2.5-5.0 M concentration of (NHa)zSOd ranged from I :128 to I :1024 by C.F. using the ‘reference serum’ PSD3. Each fraction of the scolex extract, ultracentrifuged on sucrose gradient, showed a different antigenie profile, when examined by C.F. (Fig. 1). Also the antigenic profiles of the hydatid fluid fractions were different: the 0.8-l .6-2.5-5.0 M precipitates showed antigenic peaks, by C. F., in different fractions obtained by ultracentrifugation on sucrose gradients (Fig. 2). Comparing the antigenic profiles, obtained by C.F., of the scolex and hydatid fluid fractions, they appeared quite different: the precipitates at equimolar ammonium sulphate concentration showed antigenic peaks, by C.F., in different fractions obtained by ultracentrifugation on sucrose gradients. The position of the antigenic peaks, revealed at the sucrose gradient ultracentrifugation of each precipitated fraction and characterized by the refractive index. was consistently reproducible (Fig. 3).
r-7 __________ 0.8
----l6M -
FIG. 1. Profiles, by complement
8.
M
25M
fixation, of three different scolex fractions separated on linear sucrose gradients. ‘n’ is the refractive index.
I.J.P.
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E. granulosus: antigens in scolices and hydatid fluid CF
Fraction
FIG. 2.
Profiles, by complement fixation, of three different hydatid fluid fractions separated on linear sucrose gradients. ‘n’ is the refractive index.
Identity was shown, by I.D. and I.H.A. inhibition tests, among antigens present in different scolex fractions separated by salting out and in different peaks of the same precipitated fraction observed at TABLE
A
NO
sucrose gradient ultracentrifugation (table 1). Comparable results were obtained examining the hydatid fluid fractions. Antigenic non-identity was found, when ammo-
I-CROSS-REACTION BY I.D.AMONG DIFFERENT SCOLEX FRACTIONSOBTAINED BY (NH,),SO, DIFFERENT PEAKS OBSERVED AT SUCROSE GRADIENT ULTRACENTRIFUGATION
0.8
--__~-
B
I II= 1.3520
I.6
M
2 n= 1.3630
3 ?I== 1.3800
1 n== 1.3620
2.5
M
2 II= 1.3710
3 iZ= I.3850
1 tZ= I.3575
PRECIPITATION AND ~~__
M
2 I II= II= I.3850 1.3610
5.0
M
2 ,I= 1.3720
3 = 1.3850
--I
(A = scolex fractions separated fugation). n = refractive index.
by (NH,),SO,
precipitation;
B = peaks observed at sucrose gradient ultracentri-
SILVIODOTTORINIand CARMELOTASS]
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C.F
1
20
15
10
5
25
Fraction No FE. 3. 1.6 M scolex (SC,) fraction was ultracentrifuged in three different experiments on linear sucrose gradient. The antigenic profiles, by complement fixation, were reproducible as far as the position of antigenic peaks is concerned. ‘n’ is the refractive index.
I HA
”
/ 4100
lnhlbltlon RBC
c
sensitized
with
scolex
I SC 5)
I
FIG. 4. The different fractions obtained by sucrose gradient ultracentrifugation of the ‘globulinic antigen’ and scolex 2.5 M precipitate (Scs) were tested for their inhibiting potency against sheep red blood ceils sensitized either with ‘globulinic antigen’ (upper) or with Scs scolex antigen (lower) by indirect hemagglutination inhibition method. No cross-reaction is apparent. ‘II’ is the refractive index.
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E. granulosus: antigens in scolices and hydatid fluid
FIG. 5. Slab-gel examination of the scolex fractions, obtained as described in ‘Materials and Methods’. St = Bovine albumin, ovalbumin and lysozym ; SCI = Pellet after 16,000 g; SC, = Supernatant after 16,000 g; SC, = 0.8 M fraction; SC, = 1.6 M fraction; SC, = 2.5 M fraction; SC6 = 5.0 M fraction. nium sulphate precipitates of the hydatid fluid were cross-reacted by I.D. and I.E.P. tests with similar ammonium sulphate precipitates of scolex extracts. The scolex fractions precipitated at 0.8-l&2.55.0 M-ammonium sulphate concentration did not inhibit the I.H.A., using the ‘globulinic antigen’ as sensitizing antigen: the results obtained with the 2.5 M (SG) fraction are shown in fig. 4. Also the reverse proved true. By I.D. and I.E.P. tests, two or three bands of the sheep serum were present in the scolex extract; while several, more evident bands were present in the hydatid fluid. The fractions of the scolex extract and hydatid fluid, obtained by precipitation with different concentrations of (NH4)tSO+ were examined by SDS polyacrilamide gel electrophoresis on slab-gel (figs. 5 & 6): every precipitated fraction from hydatid fluid and scolex extract is characterized by a different number of proteic bands and by their position. The 0.8 M scolex fraction showed a little number of bands with M.W. ranging between 30,000 and 65,000; no bands at lower M.W. was found. The other scolex fractions showed more bands than 0.8 M
fraction (fig. 5).
and
bands
DISCUSSION
of lower
M.W.
were
found
AND CONCLUSIONS
The antigenic profiles, by CF., of scoleces and hydatid fluid fractions, obtained on sucrose gradient ultracentrifugation, revealed a considerable molecular and antigenic heterogenicity. These profiles were consistently reproducible. The method involving ammonium sulphate salting out and subsequent sucrose gradient ultracentrifugation appears to be standardized enough to permit to ‘classify’ operatively, the hydatid antigens. A few antigenic identities between different peaks were demonstrated by the immunodiffusion test. Scolex and hydatid fluid antigens showed no identity reaction among them by immunodiffusion and indirect hemagglutination inhibition tests. This seems to demonstrate a different antigenic composition of scolices and hydatid fluid: one can suppose that antigens of hydatid fluid derive from germinative membrane and not from scolices; this is consistent with our previous observations (Pauluzzi et al.,
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SILVIO DOTTORINIand CARMELOTASSI
FIG. 6. Slab-gel examination
of the hydatid fluid ‘in foto’ and various fractions creasing ammonium sulphate concentration. St Bovine albumin and ovalbumin: AI ~~Hydatid fluid in toto; A, 0.8 M fraction ; A3 1.6M fraction ; A, -: 2.5 M fraction; A, 7 5.0 M fraction,
1977); 0.8 M fraction from scolices is immunogenic against the peritoneal secondary hydatid disease in mice, while usually this does not occur with the 0.8 M fraction from hydatid fluids: ‘functional’, according to Soulsby (1963), antigens would therefore be contained only or prevalently in scolices, not in hydatid fluid. Some antigens of sheep serum were found in scolex extracts. Hydatid materials are collected just after the slaughter of the animals and scolices are separated from hydatid fluid and washed; this would indicate that usually (not postmortem) the host proteic materials enter the parasite and are absorbed (and perhaps metabolized) into scolices. It would be necessary to ascertain if these materials are metabolized into amino-acids or if they are used to form complex proteic structures of the parasite. By slab-gel electrophoresis we have compared the proteic composition of scolex and hydatid fluid fractions and now it will be very interesting to characterize the antigens in the 0.8 hf scolex fraction. Our further efforts will be turned towards characterizing physically, chemically and antigenic-
ally this scolex importance.
fraction
obtained
because
by in-
of its biological
Acknowledgements-The authors acknowledge lent technical assistance of Mr. G. Valigi.
the excel-
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