Fractionation and characterization of hydatid fluid antigens with identification of an antigen similar to human serum albumin

TRANSACTIONS or THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE (1984) 78, 821-826 821

Fractionation and characterization of hydatid fluid antigens with identification of an antigen similar to human serum albumin

MARIO PEZZELLA~, CLAUDIO GALLI, SALVATORE DELIA, VINCENZO VULLO, FRANCESCO ZENNARO, *EUGENIO LILLINI AND FRANCO SORICE

Istituto Malattie Infettive, Universitir delgi Studi di Roma “La Sapienza”, Rome, Italy; *Istituto Zooprojilattico Sperimentale delle Regioni Lazio e Toscana, Rome, Italy

Summary Human and sheep hydatid fluids were separated by ultrafiltration, gel chromatography and

immunoabsorption into several immunogenic fractions in which both parasite antigens and host substances were present. The immunological characterization of proteic antigens was carried out by immunodiffusion and immunoelectrophoresis with rabbit and ram antisera. A line of identity was observed between a human fraction (labelled as III) and a sheep fraction (labelled as 2B). Further evidence of the presence of a parasitic antigen in fraction III was given by its reaction against an antiserum from ram directed against sheep fraction 2B. The immunological characterization of fraction III indicated a close similarity between human serum albumin and parasitic antigens.

Introduction Hydatidosis caused by Echinococcus granulosus is a

parasitic disease prevalent in Central and Southern Italy. The epidemiology and pathogenesis are known (BIANCHINI et al., 1983) but the molecular composi- tion of hydatid fluid is still unclear. The most interesting questions concern the purification of specific parasite antigens. Since in both human hydatid fluid (HHF) and sheep hydatid fluid (SHF) parasite antigens as well as host components are present, the main problem is the separation of the components of the two species. Although a great deal of immunological work has been carried out on E. granulosus antigens (CHORDI & KAGAN, 1965; BOUT et al., 1974; ORIOL et al., 1971; VARELA-D~AZ et al., 1974; POZZUOLI et al., 1975; ORIOL & ORIOL, I975), surprisingly few studies have been made on the comparative biology of hydatid fluids from different hosts. With increasing evidence of different strains of E. granulosus occurring in different areas of the world (some of which may or may not be infective to man) (SMYTH, 1977) studies on the comparative immunology of hydatid fluids from different hosts take on a new urgency.

The aim of our investigation was to characterize immunologically the proteinic antigens of HHF and SHF. In view of the established presence of “host antigens” in hydatid fluid (parasite antigens which cross-react with host components) we focused our attention on an immunogenic fraction obtained from HHF containing parasite antigens which showed a similarity with human serum albumin.

Materials and Methods Hydatid fluids

A sample of 300 ml of crude SHF from fertile liver cysts of E. granulosus was centrifuged for 30 min at 8,000 r.p.m. in an I.E.C. B20 centrifuge, rotor 870, in order to separate solid material; the supernatant was sterilized on a 0.22 pm Millipore filter. The liquid was then concentrated ten-fold at room temperature by rvaporation in a dialysis membrane (Visking). A sample o F 120 ml of HHF, obtained from fertile liver cysts of E. grun&sus, was concentrated four-fold by pervaporation and sterilized.

Gel filtration A sample of 15 ml of ten-fold concentrated SHF was

aoulied to a chromatozrauhv column (Pharmacia K26/70) packed with Sepharose 4B _ (Pharmacia) and eluted witk phosphate buffered saline (PBS 0.15 M pH 7.2), at a constant flow rate of 30 ml/h. The eluate was collected in fractions of 4.9 ml each under continuous monitoring at 280 run with an Uvicord II (LKB) apparatus. Three absorption peaks, labelled 1; 2,3, were collected. Fraction 2 was then chromatographed on Ultrogel AcA34 (range: 20,000 to 350,000): three fractions, labelled 2A, 2B, 2C, were thus obtained. Fraction 3 was further separated by ultrafiltration (Amicon cell model 202 with PM 30 membrane) into fractions 4 and 5; the former was then chromatographed on Ultrogel AcA54 (range: 5,000 to 70,000) to give fractions 4A and 4B. All SHF fractions were concentrated and dialysed to 15 ml on an Amicon Diaflo UM 2 ultra6lter. The whole procedure for the preparation of SHF fractions is summarized in Fig. 1. A samule of 15 ml of four-fold concentrated HHF was-applied to a Pharmacia K26/70 chromatography column packed with Ultrogel AcA34. The successive steps are the same as already described for SHF fractions. Four fractions (I, II, III, IV) were obtained.

Immunoabsorption Antisera: anti-sheep hydatid fluid (ASHF), anti-sheep

serum (ASS) and anti-human hydatid fluid (AHHF) were obtained by hyperimmunizing three rabbits (2.5 to 3.0 kg). The antiserum against fraction 2B of SFH was obtained from a one-year-old ram by five successive inoculations of 3 ml each of fraction 2B emulsified with 3 ml of Freund’s complete adjuvant, given at an interval of 12 days. The antiserum was collected 20 days after the last injection. The rabbit anti-sheep serum and a monospecific anti-human albumin serum from goat were insolubilized with Ultrogel AcA34, activated with gluteraldehyde, according to the method of BOSCHETTI & DELAY (1978). 15 ml of insolubilized ASS were treated separately with equal volumes of the two SHF fractions (2B and 4B) which showed immunogenic activity, and then put under mild agitation for 18 hours at 20°C. The immunosorbent was then put on a chromatography column and washed with PBS until the optical density at 280 mn was less than 0.05. Two different fractions were collected from both 2B and 4B. The fraction 2B was

tAddress: Dr. Mario Pezzella, via Silvio Pellico 42, 00195 Rome, Italy.

822 FRACTIONATION AND CHARACTERIZATION OF HYDATID FLUID ANTIGENS

separated into fractions 2BpH7, composed by all the molecules not linked to ASS, and 2BpH3, composed of the molecules linked to ASS and eluted with 0*2M glycine-HCl buffer pH 3.0. Using the same method we obtained the fractions 4BpH7 and 4BpH3 from fraction 4B.

15 ml of fraction III of HHF were added to 15 ml of Ultrogel-insolubilized anti-human albumin from goat (Behring): the same procedure used from SHF fractions was employed and we obtained fractions IIIpH7 and IIIpH3.

Immunoabsorption was not performed until the complete exhaustion of antigenic fractions because it was not necessary for the purpose of this study.

Immunodiffusion (ID) A double diffusion technique carried out in 1% agarose

O.OlM barbital buffer pH8.6 was employed (OUCHTERLONY, 1958). The plates were evaluated after an incubation of 24 to 48 hours in a moist chamber at room temperature.

Immunoelectrophoresis (ZEP) IEP was performed according to SCHEIDEGGER (1955) with

Immunoelectrofihn (Kallested) with 0.065M barbital buffer pH8.6 and a potential of 220 V at 40mA was applied for 30 min. The slides were stained with Coomassie Brilliant Blue. All the IEP reactions were carried out under the same conditions and thus the zone of electrophoretic migration in different slides was comparable.

Immunological characterization All the fractions obtained from SHF and HHF were

immunologically characterized by ID and IEP. SHF and its fractions were tested against ASHF and ASS to evaluate the immunogenicity and to check the presence of host components, and against AHHF for the presence of common antigens with HHF. HHF and its fractions were tested against AHHF, ASHF, to evaluate common antigens, and against anti-human serum (Behring) to detect the presence of host components. The fractions which reacted against anti-human serum were also tested against a monospecific anti-human albumin antiserum (Behring). The antiserum anti-2B was challenged against total sheep serum, to exclude any reactions against host components, against SHF and HHF to determine parasite antigens and against fractions IIIpH3 to confirm the presence of specific parasitic antigens.

Sepharose 48 column

/ Amicon PM 30

Ultrogel AcA 34

Ultrogel x AcA 54

/zJ @ q q Fig. 1. Preparation of S.H.F. fractions. x fractions not studied; 0 immunogenic fractions in rabbit; q not immunogenic fractions in rabbit;

Results Filtration on Ultrogel and Sepharose 4B was

performed twice for each fraction, using 15 ml each time. The elution profiles for each gel filtration resulted perfectlv comparable and similar for both HHF and SHF -fractions.

SHF profiles from Sepharose 4B showed three absorption peaks; the first one, which was composed of mu&es and other high molecular weight substances, was not studied further and was well separated ‘from the other two peaks, which overlapped slightly. The elution profile of fraction 2 on Ultrogel AcA34 showed three peaks, labelled 2A, 2B and 2C. Fraction 4 showed two peaks, labelled 4A and 4B, after Ultrogel AcA54 gel filtration.

The elution profile of HHF from Ultrogel AcA34 showed four absorption peaks, labelled I, II, III, IV, respectively.

Onlv fractions 2B and 4B from SHF showed immunogenic activity when tested by ID against ASHF. Fractions I, II and III reacted on the ID test against AHHF. Host components were found in both SHF and HHF by ID.

The anti-fraction 2B antiserum from the ram gave no precipitin arcs when tested against total sheep serum with IEP. One arc in the alpha zone appeared when this antiserum was tested against SHF and two arcs against HHF.

Fractions 2B and 2BpH7 showed four precipitin arcs with IEP against ASHF, while only three of these arcs were detectable when these two SHF fractions were assayed against ASS: two and one precipitin arcs, respectively, were produced against AHHF. Fractions 2BpH3 gave the same three arcs against both ASHF and ASS, while two thin arcs were detectable against AHHF (Tabel I).

Fractions 4B and 4BpH3 showed two precipitin arcs, one in the alpha and one in the beta zone, against

Table I-Immunoelectrophoresis of SHF fractions against anti-sheep hydatid fluid (ASHF), anti-sheep serum (ASS) and anti-human hydatid fluid (AHHF) from rabbit. Indication of number of precipitin arcs

ASHF ASS AHHF

Table II-Immunoelectrophoresis of HHF fractions against anti-sheep hydatid fluid (ASHF), anti-human hydatid fluid (AHHF), anti-human serum (AHS) and anti-human albumin (AHA) from rabbit. Indication of number of precipitin arcs

ASHF AHHF AHS AHA

I 1 2 1 0 ::I : : 2 3 1

IIIpH3 1 1 1 :

M. PEZZELLA et al.

A. In troughs A and B: anti-human hydatid fluid.

B. In troughs D and E: anti-human albumin.

823

C. In trough F: anti-human serum.

D. In troughs E and F: anti-fraction 2B.

Fig. 2. IEP of fraction IIIpH3 in well 2, 5,7, 6. An albumin-zone precipitin arc of similar shape is evidenced against anti-human hydatid fluid, anti-human albumin, anti-human serum and anti-fraction 2B antiserum.

Fig. 3. In well 1A and 1B: fraction 2B; in well 6A and 6B: fraction III; in well 7A: anti-sheep hydatid fluid; in well 7B: am&homao hydahd fluid. The two fractions III (from HHF) and 2B (from SHF) gave an image of identity.


A. In troughs C and D: anti-human hydatid fluid.

B. In troughs C and D: anti-human serum.

C. In troughs C and D: anti-sheep hydatid fluid.

Fig. 4. IEP of fraction III in well 4A, 4B, 4C. The smaller arc in albumin zone (arrow, photo A and C) appears to be formed by a parasitic component since it is detectable against anti-sheep hydatid fluid and anti-human hydatid fluid and not against anti-human serum. The bigger arc (double arrow, photo B) is constituted by human serum albumin.

Fig. 5. Migration differences between human hydatid fluid and fraction 2B from sheep hydatid fluid. In troughs C, D, E: anti-sheep hydatid fluid; in well 4: fraction 2B; in well 5; human hydatid fluid.

both ASHF and ASS. An alpha-zone precipitin arc appeared also against AHHF. Fraction 4BpH7 always gave an alpha-zone precipitin arc, whatever the antiserum employed (Table I). Fraction 1 of HHF showed a large and shaded precipitin arc, which spread from alpha zone to the well, at IEP against ASHF, ASS and AHHF. Another alpha-zone arc was produced against AHHF (Table II).

Fraction II of HHF gave a single precipitin arc in the alpha zone when tested at IEP against AHHF, ASHF, anti-human serum and anti-human albumin. A second arc, lying in the beta zone, was produced only against anti-human serum (Table II).

Fraction III of HHF gave an albumin zone precipitin arc against AHHF, ASHF, anti-human serum and anti-human albumin (Table II). A further arc in the albumin zone appeared against AHHF, while two and one alpha-zone precipitin arcs were

evident against anti-human serurh and anti-human hydatid fluid, respectively (Fig. 4).

Fraction IIIpH3 (eluted from insolubilized monospecific anti-human albumin) formed an albumin-zone arc of similar shape against AHHF (Fig. 2A), anti-human albumin (Fig. 2B), anti-human serum (Fig. 2C) and anti-fraction 2B (Table II).

Fractions III and 2B gave an image of identity with ID against AHHF and ASHF (Fig. 3). Migration differences between HHF and SHF proteic antigens were apparent by the IEP reaction of HHF and fraction 2B et IEP (Fig. 5).

Discussion The use of ultrafiltration, gel chromatography and

immunoabsorption allowed us to separate from human and sheep hydatid fluids several fractions, each

M. PEZZELLA et al. 825

of which is composed of various molecular species with different biological characteristics.

By immunoabsorption we identified in sheep hydatid +ltid fraction 2B a precipitin line due only to parasite antigens. The parasite nature of this arc was established by its presence when fractions 2B and 2BpH7 were assayed against anti-sheep hydatid fluid and by its absence when the reaction was performed against anti-sheep serum. No precipitin arcs due to parasite antigens alone were evident in fraction 4B, since fractions 4BpH7 and 4BpH3 gave the same electrophoretic pattern against both anti-sheep hydatid fluid and anti-sheep serum. In the meantime, the reaction against anti-human hydatid fluid confirmed the presence of parasite antigens in fraction 4B.

In order to avoid any reactions against host components we prepared a sheep antiserum directed against fraction 2B. The lack of reactivity against total sheep serum and the strong reaction against both sheep and human hydatid fluids at IEP controls confirmed that this antiserum is directed against substances of certain parasite origin only.

We studied fraction III of human hydatid fluid thoroughly, since it gave an image of identity with fraction 2B and ID against both anti-sheep hydatid fluid and anti-human hydatid fluid. This fraction gave two arcs in the albumin zone at IEP against anti- human hydatid fluid, and a precipitin arc quite similar to albumin against anti-human serum (double arrow, photo 4B). This latter arc is probably formed by human serum albumin, while the smaller one could be formed by parasite substances, since it is not detect- abzr;Fe;t ar&GHy ser;n&arrow~~ig. 4A, 4C).

Ultrogel- insolubilized anti-h&an albumin, showed an albumin-zone arc at IEP against anti-human hydatid fluid, anti-human serum, &-&human albumin and anti- fraction 2B. This arc, morphologically identical in all reactions, indicates in our opinion that human serum albumin is strictly related to a parasite antigen in human hvdatid fluid. The reactions of human hvdatid fluid and-sheep hydatid fluid and their fractions were carried out to identify the precipitin lines due to parasite components only, whereas the arcs shown against autologous antisera reflected the presence of b;th host a6.l parasite substances. A‘ migration difference was noted between human hvdatid fluid parasite arc, which lays in the albumin zbne, and 2B (sheep hydatid fluid) parasite arc, which is detectable in alpha- 1 zone (Fig. 5).

This difference mav be due to different strains of E. granulosus in differeni hosts, as suggested by McMA- NUS & SMYTH (1979); common antigens should, however, be present in the various strains. On the other hand, if the same strain is present in both man and sheep, the parasite antigens could be chemically different in two species. A possible explanation of this finding may be the adaptation of parasite metabolism in different hosts. Thus, the same parasite antigen can be included in different molecular structures, with a diverse electrophoretic behaviour. Another possible explanation of ihe differences between sheep-hydatid fluid and human hvdatid fluid mav be the inclusion of host and parasite molecules in stable molecular complexes,which would protect the parasite from the immune response of the host. This hypothesis is further supported by the strict association between a

parasite antigen and human serum albumin in fraction III pH3 of human hydatid fluid. At this point we cannot exclude any of these hypotheses, even though the last one seems to have some sunnort from our findings.

. .

It is evident that the parasite antigens isolated in the past and differently named as “antigen 4”, “antigen 5” (CHORDI & KAGAN, 1965; POZZUOLI et al., 1975), “antigen A”, “antigen B” (ORIOL et al., 1975), “antigen 5” (CAPRON et al.. 1970: VARELA-D~AZ et al., i974) d& not constituie the’ total of parasite antigens. All the former authors separated host components completely before isolating parasite antigens, and immunological reactions with these result- ing antigens were often less effective than those performed with total hydatid fluid. Our data indicate that the parasite antigens of hydatid fluid may be similar to host antigens; thus, the elimination of host components could lead to the loss of immunoreactive parasite substances.

We suggest a different approach to the study of E. granulosos antigens, employing heterologous antisera in order to separate the parasite components of hydatid fluid. This would give better results in the way of isolating all parasite antigens and would represent the first and most important step as far as the immunological diagnosis of hydatidosis is con- cerned.

References Bianchini, C., Sanguigni, S., RUSSO, V. & Ilardi, I. (1983).

Epidemiologia delle ehnintiasi in Italia. Gtimale di Malattie Znfettive e Parassitarie, 35, 1193-1199.

Boschetti, E. & Delay, M. (1978). Gluteraldehyde-activated Ultrogel polyacrylamide gel in the purification of trypsin by affinity chromatography. Science Tools? 25, 18-31.

Bout, D., Fruit, J. & Capron, A. (1974). Purdication d’un antigltne sptcifique du liquide hydatique. Annales d’lm- munologie (Znstitut Pasteur), 125 C, 775-788.

Capron, A., Yarzabal, L., Vernes, A. & Fruit, J. (1970). Le diagnostic immunologique de l’khinococcose humaine (bilan personnel B propos de 400 observations). Patholo- gie Biologique, 18, 357-365.

Chordi, A. & Kagan, I. (1965). Identification and characterization of antigenic components of sheep hydatid fluid by immunoelectrophoresis. 313umal of Parasitology, 51, 63- 71.

McManus, J. P. & Smyth, J. D. (1979). Isoelectric focusing of some enzymes from Echinococcus granulosus (horse and sheep strains) and E. ~ultilocularis. Transactions of the ~5~~lSociety of Tropzcal Medicine and Hygiene, 73,

Oriol, R., kllkuns, J. F., Perez-Esandi, M. V. & Oriol, C. (1971). Purification of lipoproteic antigens of Echinococ- cus granulosus from sheep hydatid fluid. AmericanJournal of Tropical Medicine and Hygiene, 20, 569-574.

Oriol, C. & Oiiol, R. (1975). Physicochemical properties of a lipoprotein antigen of Echirwcoccus granulosus. American Journal of Tropical Medicine and Hygiene, 24, 96-100.

Ouchterlony, 0. (1958). Diffusion in gel methods for immunological analysis. Progress in Allera, 5, l-58.

Pozzuoli, R., Piantelli, M:, Perucci, C., Arru, E. & Muslani, P. (1975). Isolation of the most immunoreactive antigens of Echinococcus granulosus from the sheep hydatid fluid. Journal of Immunology, 115, 1459-1463.

Scheidegger, J. S. (1955). Une micro-mkthode de l’immu- notktrophortse. International Archives of Allergy, 7, 103-110.


Smyth, J. D. (1977). Strain differences in Echinococcus grundosas? with special reference to the status ,of equine hydatidosis, in the United Kingdom. Transacti? of the f3ylah Socwty of Troprcal Medrcrne and Hygme, 71,

Varela-D&z, V. M., Coltorti, E. A., Ricardcs, M. I. & Guisantes, G. A. (1974). The hnmunoelecuophoretic

characterization of sheep hydatid cyst fluid antigens. American Jounurl of Tropical Medicine and Hygiene, 23, 1092-1096.

Accepted for publication 24th February, 1984

New Fellows Elected 18th October, 1984 (continued)

Continued from p. 789 Pribadi, W., Indonesia Pyne, C. E. J., Britain Rahman, M. U., Pakistan Raj, D., India Raijuddin, S. K., India Rischette, Rene, Luxembourg Rizzi, M., Italy Rosario, V. E., USA Roscigno, G., Switzerland Roux, J. F., Tahiti Roy, A. K., India Saghari, H., Iran Sagua, H. F., Chile Saha, M. N. N., India Saha, S. G., India Sahaj S., India Saha. S.. India Salagky,’ USA &manta, B. K., India Sannigrahi, S., India Saravia, Nancy G., Colombia Sarkar, B. K., India Schapira, A., Mozambique Sen, N. K., India Shears, P., Britain Shinondo, C. J., Zambia

Sidhu, P., Britain Smith, Marie A., Britain Soleimani, Z., Iran Sowah, S. A., Togo Striebel, H. P., Switzerland Sullivan, J. B., USA Sumarmo, Indonesia Sutanto, I., Indonesia Sutton, C. W., Britain Thakur, C. P., India Titanji, V., Cameroon Toure, Y. T., Mali Unandar, B., Indonesia Vedamanickam, R., India Vetter, J. C. M., Netherlands Wardhan, H., India Washino, R. K., USA Watt, G., USA Weiss, L. M., USA Weller, R. D., USA Williams, G. V:, Australia Wilson, G., Britain Wilson, Mary E., USA Wulur, J. H., Indonesia Yang, R., Upper Volta

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Fractionation and characterization of hydatid fluid antigens with identification of an antigen similar to human serum albumin