Comp. Biochem. Physiol. Vol. 85B, No. 3, pp. 627-633, 1986 0305-0491/86 $3.00 + 0.00 Printed in Great Britain Pergamon Journals Lid

PHOSPHORYLCHOLINE BEARING COMPONENTS OF SOME HELMINTHIC PARASITES:

LOCALIZATION TO PARASITE LIPOPROTEINS

A. S. McWILLIAM*, G. A. STEWART t and W. ALLEN Clinical Immunology Research Unit, Princess Margaret Hospital, Thomas Street, Subiaco,

Western Australia 6008 (Tel: 382-8592)

(Received 27 January 1986)

Abstract--l. Extracts of various helminthic parasites were studied to determine the molecular carrier of the hapten phosphorylcholine (Pc).

2. Physicochemical studies showed that the carrier was heterogeneous with regard to apparent molecular weight (70,000-> 106 daltons).

3. Ultracentrifugal analysis of Ascaris suum and Nippostrongylus brasiliensis showed that Pc was associated with low (LDL), high (HDL) and a heterogeneous group of very high density (VHDL) lipoproteins.

4. The majority of Pc was found in the most dense VHDL (d > 1.25 gml ~). 5. The apparent molecular weight and particle diameter of A. suum HDL was 751,000 daltons and

16.2 nm, respectively. 6. The apparent molecular weights and particle diameters of A. suum VHDL ranged from 469,000 to

677,000 daltons and 13.6 to 15.5 nm, respectively. 7. The electrophoretic mobilities of A. suum LDL and HDL were /3 and 7, respectively. 8. A. suum VHDL was electrophoretically heterogeneous. 9. It is probable that Pc is associated with the phospholipid phosphatidylcholine.

INTRODUCTION

While the hapten phosphorylcholine (Pc) has been found in extracts of many helminthic parasites such as Nippos t rongy lus brasiliensis, H a e m o n c h u s con tor - tus, Ascar is suum, Nematosp i ro ides dubius, D i c t y o - caulus viviparus and D. f i lar ia (Pery et al., 1974; Potter, 1970; Pery, 1977) its biochemistry is unclear. Despite this, however, Pc has been of interest because of the anti-Pc immune response it stimulates in hosts infected with Pc containing parasites. For example, mice infected with either A. suum or N. brasiliensis, or injected with the Pc containing pneumococcal C-polysaccharide produce high levels of Pc specific antibody (Crandall and Crandall, 1967, 1969, 1971; Mitchell et al., 1976a,b).

On the basis of these observations, the potential of Pc-containing material for use as vaccines has been investigated, not only for parasitic disease but also for bacterial disease. In this regard, Pery et al. (1975) showed that subcutaneous injection of rats with a Pc containing putative C-substance from N. brasil iensis injected 8 days prior to inoculation with viable N. brasil iensis larvae resulted in a significant reduc- tion in intestinal worm burden in rats. With regard to bacterial disease, Briles et al. (1981) showed that the presence of anti-Pc antibody in normal mouse sera

*Present address: Plant Pathology Laboratory, Western Australian Department of Agriculture, Baron-Hay Court, South Perth, Western Australia 6161.

tAuthor to whom correspondence should be addressed.

protected against intravenous infection with WU2 type 3 Strep tococcus pneumon iae and, in addition, that mice unable to produce anti-Pc antibody were susceptible to infection with these bacteria.

While Pc is found in phospholipids and in phos- pholipid moieties of vertebrate lipoproteins, and in bacterial and fungal C-substances, the molecular localization of the Pc containing moiety in parasites such as A. suum is unknown. However, some studies have suggested that the parasite Pc carrier molecule was analogous to pneumococcal C-substance (Pery et al., 1979; Fletcher et al., 1980) but this was not conclusively demonstrated. The possibility that Pc is associated with parasite lipoproteins has not yet been investigated.

In this report, we quantitate Pc in several parasite extracts using a sensitive haemagglutination- inhibition assay and examine the molecular weight distribution of the Pc bearing moieties in selected parasite extracts in an attempt to determine the molecular localization of this hapten. Further, we describe the isolation of several lipoprotein classes from A. suum perienteric fluid and an adult worm extract of N. brasiliensis. In addition, we examine parasite extracts for reactivity with the lectin Con A since, previous studies have shown a structural cor- relation between the presence of Con A reactive sugar residues and Pc (Cifonelli et al., 1966; Baldo et al., 1977).

MATERIALS AND METHODS

Parasite extracts

Ascaris lumbricoides worms were collected in Indonesia

627

628 A.S. McWILLIAM et al.

through the courtesy of members of the Department of Parasitology, University of Indonesia. Toxocara canis worms obtained from dogs on autopsy were kindly provided by Associate Professor Turner.

Nippostrongylus brasiliensis worms and larvae were ob- tained from infected WAG rats housed in our laboratory. Strongyloides ratti L3 larvae were provided by Dr H. Dawkins, Department of Medicine, University of Western Australia. Schistosoma rnattheei were the gift of Dr E. Fourie, Department of Microbiology, University of Pre- toria, Republic of South Africa.

Soluble extracts of these parasites were prepared in 0.01 M phosphate buffer, pH 7.2, containing 0.15 M NaC1 (PBS) using a Potter-Elvehjem tissue grinder. Insoluble material was removed by centrifugation and the extracts stored at -20°C. N. brasiliensis larvae and secretory/ excretory extracts were prepared as described by Allan and Mayrhofer (1981). Ascaris suum perienteric fluid was isolated from worms obtained from a local abattoir.

The protein content of each extract was determined with the modified Folin assay (Lowry et al., 1951).

Preparation o f S107 serum

The S107 anti-phosphorylcholine mouse myeloma (Pot- ter, 1970) was obtained from Dr N. Warner of the Walter and Eliza Hall Institute, Melbourne. The myeloma was passaged by subcutaneous injection in BALB/c mice and the serum pooled, filtered and stored at -20°C.

Preparation o f phosphorylcholine (Pc )-bovine serum albumin conjugate (Pc-BSA )

Para-diazonium phenylphosphorylcholine was prepared from p-nitrophenyl-phosphorylcholine (Biosearch, San Rafael) according to the method of Chesebro and Metzger (1972). This material was coupled to bovine serum albumin (BSA, Sigma Chemical Co., Ltd, USA) as described by Kishimoto et al. (1979). The molar coupling ratio of Pc to BSA was shown to be 11:1 using a molar absorbency of 12,600 at 475nm in 0.1N NaOH.

Gel filtration studies

The parasite extracts were fractionated on a column (81 cm × 2.6 cm) of Sephaeryl S-300 (Pharmacia South Seas Pty. Ltd, Australia) equilibrated with 0.05 M ammonium bicarbonate buffer, pH 8.0. Fractionation was achieved by upward elution at 20mlhr -~ and 3 ml fractions were collected. Elution profiles were determined by measuring the absorption at either 280nm or 230nm. Appropriate fractions were pooled and lyophilized.

The apparent molecular weight distribution of the Pc containing components in the various parasite extracts were obtained by reference to a standard curve of K,v versus log molecular weight. The apparent molecular weight markers were obtained from Pharmacia (South Seas) Pty. Ltd, Australia and included thyroglobulin (669,000 dal- tons), ferritin (440,000 daltons), catalase (232,000 daltons), aldolase (158,000 daltons), bovine serum albumin (67,000 daltons), ovalbumin (43,000 daltons), chymotrypsinogen (25,700 daltons) and ribonuclease (13,700 daltons). Data were analysed by linear regression.

Detection o f phosphorylcholine containing components

The presence of Pc was determined by an inhibition of haemagglutination (HA1) assay using a Pc specific anti- serum obtained from mice bearing the S107 plasmacytoma and sheep red blood cells coated with an extract of A. suum perienteric fluid as described previously (Stewart and Turner, 1980). Results were expressed in haemagglutination inhibition units (HAIU) where one unit was the amount of test substance in #g of protein required to inhibit the agglutination of sensitized cells by the $107 serum. Specific activity was expressed as HAIU per mg inhibitor protein.

Detection o f Con A reactive components

Con A reactive components were detected by a HA1 assay using guinea pig red blood cells (Smith and Walborg, 1976) in a manner analogous to that described for Pc-containing components. Results were expressed in HAIU as described above.

Preparative ultracentrifugation

Ultracentrifugation experiments were performed in a Beckman Model L ultracentrifuge at 14°C using a Ti 50 rotor employing experimental conditions commonly used in the isolation of human lipoprotein classes. Whilst the re- lationship between parasite and human lipoprotein classes is unknown, the terminology used to describe the latter has been adopted throughout this study. Low density lipo- proteins (LDL) were isolated at 140,000g for 24 hr using a NaCI-KBr solvent of density 1.063 g ml -~. After centrifu- gation, the top 2 ml fraction from each centrifuge tube was removed and the density of the remaining solution adjusted to 1.210 g ml -~ by the addition of solid KBr. High density lipoproteins (HDL) were isolated at this density by centrif- ugation at 140,000g for 40hr after which the top 2ml fraction from each tube was removed. The density of the remaining solution was adjusted to 1.250gml -~ by the addition of solid KBr and the very high density lipoprotein (VHDL) isolated by centrifugation at 165,000g for 44hr. All ultracentrifuge fractions were dialyzed against 0.15 M NaCI containing 0.01 M EDTA for 48 hr at 4°C.

Electrophoretic methods

Agarose gel electrophoresis (AGE) was performed using 1% w/v agarose (Type 5, Sigma Chemical Co. Ltd, USA) in barbitone buffer, pH 8.6 (I = 0.05). Samples were pre- stained with Sudan Black B (Narayan, 1975) and electro- phoresed at 6Vcm -~ for 1 hr at 4°C. Gels were photo- graphed immediately after completion of electrophoresis or fixed in picric acid and stained with Coomassie blue R250.

Gradient polyacrylamide gel electrophoresis (GPAGE) was performed at 20°C using the Pharmacia (South Seas) Pry. Ltd gel electrophoresis apparatus (GE-4) and pre- poured gradient gels, either 4-30% (Pharmacia) or 2.5--27% (Gradipore, Sydney, Australia). Electrophoresis was per- formed according to the manufacturer's instructions. Molecular weight (mol. wt) markers were purchased from Pharmacia (South Seas) Pry. Ltd and included thyro- globulin (mol. wt: 669,000) ferritin (440,000), catalase (232,000), lactate dehydrogenase (140,000) and albumin (67,000). Molecular weight determinations were performed in triplicate and the data analysed by linear regression.

The particle diameters (in nm) of A. suum lipoproteins were determined in a manner similar to that described for the characterization of human high density lipoproteins (Blanche et al., 1981) using the above molecular weight reference proteins. The particle diameters used in the calcu- lation were 17.0, 12.2, 10.4, 8.2 and 7.1 nm, respectively. The data were analyzed by linear regression analysis.

Samples were prestained for lipoprotein with Sudan Black B (Narayan, 1975) and photographed immediately after electrophoresis. Gels were subsequently stained for protein with Coomassie blue G250 (Blakesly and Boezi, 1977) and for carbohydrate with the periodic acid-Schiffs reagent. Preparative GPAGE was also performed on the VHDL fractions (d > 1.25 g ml- ~ ) from A. suum and N. brasiliensis to determine the molecular weight distribution of Pc. After electrophoresis of these fractions, gels were sequentially sectioned and extracted with PBS. The resulting fractions were analyzed for Pc by HAl.

RESULTS

Pc content o f parasite extracts

The presence of Pc in the var ious extracts was

Parasite lipoproteins and phosphorylcholine 629

Table 1. Reactivity of various parasite extracts with the phosphorylcholine specific plasmacytoma S107 and with Con A

S107 Con A Parasite Type of extract reactivity* reactivity

Ascaris suum Perienteric fluid 315,985 46 Ascaris lumbricoides Adult whole worm 169,994 ND Toxocara canis Adult whole worm 507,834 14 Nippostrongylus brasiliensis Adult whole worm 126,732 0 Nippostrongylus brasiliensis L 3 larvae 81,541 0 Nippostrongylus brasiliensis Secretory/excretory 51,807 0 Strongyloides ratti L 3 larvae 682,666 ND Schistosoraa mattheii Adult whole worm 124 465 PC-BSA - - 1.46 × 109 ND

*Results are expressed in haemagglutination inhibition units per mg (HAIU/mg) inhibitor protein, where 1 HAIU represents the amount of inhibitor required to inhibit the agglutination of sensitized cells by the S107 plasmacytoma serum or guinea pig red blood cells by Con A.

ND = Not done.

determined using a haemagglutination inhibition as- say, the specificity of which confirmed using a Pc-BSA conjugate. These studies showed (Table 1) that while the extracts contained Pc in varying amounts, extracts of A. suum (315,985 HAIU/mg), S. ratti (682,666 HAIU/mg) and T. canis (507,834 HAIU/mg) contained the most Pc. Extracts of N. brasiliensis adult worm and L 3 larvae contained 126,732 and 81,541 HAIU/mg inhibitor protein re- spectively which were approximately 2.5- to 8-fold less than those obtained with extracts of A. suum, S. ratti and T. canis. The secretory/excretory extract of N. brasiliensis adult worms contained 1.6- and 2.4-fold less Pc than did the N. brasiliensis L 3 and adult worm extracts, respectively. The Pc content of the A. lumbricoides adult worm extract (169,994 HAIU/mg inhibitor protein) was similar to that obtained with the N. brasiliensis adult worm extract but was approximately half that of the A. suum perienteric fluid. The S. mattheei extract contained little Pc (124HAIU/mg inhibitor protein) in com- parison with the other extracts.

Con A reactivity o f parasite extracts

Of the extracts examined (Table 1), only A. suum, T. canis and S. rnattheei reacted with Con A which contained 46, 14 and 465 HAIU/mg inhibitor protein respectively. In contrast, none of the N. brasiliensis extracts reacted with Con A. By far the most potent of the extracts examined was S. mattheei which contained approximately 10- and 33-fold more Con A reactive material than did extracts of A. suum and T. canis, respectively.

Molecular weight distribution o f Pc-containing com- ponents

Various parasite extracts were fractionated on Sephacryl S-300 to determine the apparent molec- ular weight distribution of the Pc-containing com- ponents therein. These studies (Figs 1 and 2) showed that in all parasite extracts examined, Pc eluted over a wide molecular weight range. The results (using those obtained with N. brasiliensis adult worm as a basis for interpretation) suggested that each extract comprised 3 Pc-containing peaks differing in molec- ular weight. The first Pc-containing peak eluted in the void volume (mol. w t > 106 daltons). The second peak had an apparent molecular weight of 669,000

daltons whereas the third peak had an apparent molecular weight of about 200,000 daltons. The Pc profile of each extract differed substantially from the corresponding protein elution profile.

Preparative ultracentrifugation

As lipoproteins are known to contain the hapten Pc, it was decided to determine whether any of the parasite Pc was associated with these molecules. Insufficient material precluded an examination of most extracts but sufficient A. suum perienteric fluid and N. brasiliensis adult worm extract were available. These materials were subjected to sequential ultra- centrifugation at solvent densities used in the iso-

o~ A

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00 S

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Fig. 1. Sephacryl S-300 (81 cm x 2.6 crn) gel filtration anal- ysis of extracts of Toxocara canis (A), Nippostrongylus brasiliensis adult somatic (B) and Ascaris suum perienteric body fluid (12). Elution was monitored at 280 nm or 230 nm (0) for protein, and for phosphorylcholine content (m) using the Pc specific S107 serum in an haemagglutination-

inhibition assay. V 0 = 174 ml.

630 A. S. McWILuAM et al.

08 t LARVAL

o L o 0 8 SECRETORY

I J

0 8 SOMATIC

I E c

c-,

180 3OO

V OLUMk~ m

12

8

4

I 0

8 r

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Fig. 2. Sephacryl S-300 (81 cm x 2.6 cm) gel filtration anal- ysis of various extracts of Nippostrongylus brasiliensis. Lar- val extract, secretory/excretory extract and adult somatic extract. Elution was monitored at 280 or 230 nm (0 ) for protein, and for phosphorylcholine content (11) using the Pc specific myeloma S107 in an haemagglutination inhibition

assay.

lipoproteins with varying mobilities. The LDL frac- tion electrophoresed as a broad, indistinct band in the /3 region, whereas the H D L remained at the origin (7 electrophoretic mobility). V H D L fractions 1, 2, 3 and 4 all contained a lipoprotein band with a/3 electro- phoretic mobility. However, in addition, V H D L frac- tion 1 and 3 contained a lipoprotein band at the origin (7 mobility) and V H D L fraction 4 contained an ~ migrating protein.

G P A G E analysis of the lipoproteins from A. suum (Fig. 4; Table 3) showed that the LDL fraction did not enter the gel indicating that the molecular weight of this material was very high ( > 2 x 106 daltons). The H D L was shown to have an apparent molecular weight of 751,000 daltons (particle diameter of 16.2nm) whereas the apparent molecular weight of V H D L fraction 1 was 677,000 daltons (particle diameter of 15.5nm). The apparent molecular weights and particle diameters of V H D L fractions 2, 3 and 4 were 610,000 and 15.0, 535,000 and 14.3, and 469,000 daltons and 13.6nm respectively. Pre- parative G P A G E of A. suum V H D L fraction 4 and N. brasiliensis V H D L fraction 2 was also performed to determine the molecular weight distribution of Pc. These studies (data not presented) showed that distri- bution was heterogeneous in accord with the pre- viously described gel filtration studies. However, in A. suum V H D L fraction 4, one major peak of Pc was detected at a position corresponding to that of the V H D L fraction 4 lipoprotein (469,000 daltons) and a minor peak in a position corresponding to a protein of molecular weight of 70,000 daltons.

lation of human lipoproteins. LDL, HDL, V H D L fractions were obtained and tested for Pc content in a HA1 assay. The V H D L fraction of A. suum was further divided into four subfractions since a dis- tinctive banding pattern was observed after ultra- centrifugation; fraction 1 represented the VHDL, fraction 2 represented the infranatant between frac- tion 1 and a characteristic brown band situated half way down the centrifuge tube, fraction 3 represented the brown band and fraction 4 represented the red coloured solution remaining below fraction 3. This banding pattern was not observed with extracts of N. brasiliensis; fraction 1, therefore, represented the V H D L and fraction 2 represented the material sedimenting to the bot tom of the tube.

Results (Table 2) showed that while LDL, H D L and V H D L fractions from both parasites contained Pc, the majority of the Pc was located in the A. suum V H D L fraction 4 (92%) and N. brasiliensis V H D L fraction 2 (99%). Sephacryl S-300 gel filtration anal- ysis showed that the molecular weight distribution of the Pc carrier in these latter Pc rich fractions was very similar to that demonstrated by the original extracts prior to ultracentrifugation (data not presented).

Electrophoretic studies

The fractions obtained by preparative ultra- centrifugation of A. suum perienteric fluid were pre- stained for lipid with Sudan Black B and analysed by both A G E and G P A G E .

A G E studies at pH 8.6 (Fig. 3) showed that A. suum contained at least 3 electrophoretically distinct

DISCUSSION

Extracts of A. suum, A. lumbricoides, N. brasilien- sis, S. ratti, T. canis, in contrast to those of S. mattheei , were shown to contain Pc extending pre- viously reported findings (Potter, 1970; Crandall and Crandall, 1971; Pery et al., 1974; Pery, 1977).

Whilst it was not possible to analyse all devel- opmental stages of the parasites studied here, it was demonstrated that both larval, adult worm stages and

Table 2. Reactivity of lipoprotein fractions from Ascaris suum perienteric fluid and Nippostrongylus brasiliensis adult worm extracts with the phosphorylcholine specific murine plasmacytoma S107 in a

haemagglutination inhibition assay

Reactivity with S107 Recovery't Fraction total HAl* units %

A . suum

LDL 532,480 0.08 HDL 122,880 0.02 VHDL Fraction 1 6,912 0.001 VHDL Fraction 2 196,608 0.03 VHDL Fraction 3 46 × 1 0 6 7.7 VHDL Fraction 4 545 x 106 92

N. brasiliensis LDL 22,528 0.06 HDL 10.496 0.03 VHDL Fraction 1 35,328 0.10 VHDL Fraction 2 34 X 1 0 6 99.7

*Results are expressed in haemagglutination inhibition units where 1 unit represents the minimum volume of inhibitor required to inhibit the agglutination of sensitized cells by the SI07 plasma- cytoma serum.

tRecovery of Pc in each fraction is expressed as the amount of Pc in each fraction divided by the total amount of Pc in all fractions.

Parasite lipoproteins and phosphorylcholine 631

Fig. 3. Agarose gel electrophoretic analysis of Ascaris suum perienteric fluid (A), LDL (B), HDL (C), VHDL fractions 1 (D), 2 (E), 3 (F) and 4 (G) prestained for lipid with Sudan Black B.

excretory/secretory products contained Pc. The most potent Pc containing extracts were those of S. ratti, T. canis and A. suum followed by extracts of N. brasiliensis (whole worm, larval, excretory/secretory) and A. lumbricoides.

The apparent molecular weight distribution profile,

A B C D E F G

1

2

3

4

5

Fig. 4. Gradient polyacrylamide gel electrophoretic analysis of Ascaris suum perienteric fluid (A), LDL (B), HDL (C), VHDL fractions 1 (D), 2 (E), 3 (F) and 4 (G) stained for protein with Coomassie blue G250. The molecular weight markers (M) used were thyroglobulin (1, mol. wt 669,000 daltons), ferritin (2, 440,000), catalase (3, 232,000), lactate

dehydrogenase (4, 140,000) and albumin (5, 67,000).

determined by gel filtration of the Pc carrier was similar in each type of extract studied. Although the molecular weight profiles were heterogeneous, there appeared to be 3 main peaks of Pc present in each extract with apparent molecular weights of > 10 6, 700,000 and 200,000 daltons, respectively. G P A G E studies produced similar results and, in addition, suggested that a further Pc containing component was also present with an apparent molecular weight of about 70,000 daltons.

The nature of the Pc carrier in parasite extracts is unknown but two types of substances are known to contain Pc or related compounds, namely, bacterial or fungal C-substances and lipoprotein bound phos- pholipids. Thus, an attempt was made to determine whether parasite Pc was associated with either of these compounds.

Several of the extracts were examined for Con A reactivity since previous studies demonstrated that fungal and bacterial "C-substances" containing Pc contain high levels of Con A reactive sugar residues, (Cifonelli et al., 1966; Baldo et al., 1977). Whereas extracts of the Pc deficient S. mat thee i reacted strongly with Con A, in agreement with studies on other schistosomes (Boros et al., 1977; Carter and Colley, 1979), the Pc rich extracts such as A. suum, T. canis and N. brasiliensis reacted weakly or not at

Table 3. Apparent molecular weights of lipoprotein fractions isolated from Ascaris suum perienteric body fluid

Apparent molecular Particle diameter Lipoprotein fraction weight (daltons) (nm) LDL >2 x 106 - - HDL 751,000 16.2 VHDL Fraction I 677,000 15.5 VHDL Fraction 2 610,000 15.0 VHDL Fraction 3 535,000 14.3 VHDL Fraction 4 469,000 13.6

632 A.S. MCWILLIAM et al.

all. Similar negative findings with regard to Con A have been reported for N. brasiliensis using both precipitation and affinity chromatography techniques (Pery, 1977). On the basis of these data it is prob- able that parasite derived Pc is not associated with components analogous to bacterial and fungal "C-substances".

In an attempt to determine whether the parasite Pc carrier was a lipoprotein, LDL, HDL and VHDL fractions were isolated by preparative ultra- centrifugation and were tested for the presence of Pc. These studies showed that LDL, H D L and VHDL fractions from both A. suum perienteric fluid and N. brasiliensis whole worm extract contained Pc, al- though the majority of the Pc in both extracts was found in the most dense fractions (VHDL). On the basis of these data, it is probable that the Pc identified in helminthic parasite extracts is part of the phos- pholipid phosphatidylcholine, a molecule present in many helminthic parasites (reviewed by Frahya and Smith, 1983).

The major port ion of the Pc containing phos- pholipid was found in the most dense of the VHDL fractions, a situation analogous to that found on ultracentrifugal analysis of mammal ian sera. How- ever, in contrast to the mammal ian data, where the majority of the phospholipid in LDL, HDL and VHDL depleted serum was bound to a lbumin (Switzer and Eder, 1965), the majority of parasite phospholipid was bound to a lipoprotein (of apparent molecular weight 469,000 daltons) and the minority bound to an, as yet, unidentified carrier of apparent molecular weight of 70,000.

The lipoproteins isolated from A. suum, particu- larly the HDL and VHDL appear to differ from their mammal ian counterparts not only with regard to molecular weight but also with regard to electro- phoretic mobility. For example, in mammal ian sera, H DL and VHDL migrate as ct proteins but A. suum H DL migrated as a 7 protein. Further, A. suum VHDL migrated as a fl protein and, depending on density, together will either a ~ protein or an ct protein. In addition, it is clear that the particle diameter of A. suum HDL (16.2 nm) is higher than that of its mammal ian counterpart which for example, ranges from 8.5 to 12.0 nm (Anderson et al., 1977).

The available data on lipoproteins from other helminthic parasites is limited but several lipoproteins have been isolated from Echinococcus granulosus and Fasciola hepatica (Oriol et al., 1971; Korach, 1966). The relationship between the lipoproteins from A. suum and the above parasites is unknown but it is possible that they may be similar. For example, it has been shown that the lipoprotein designated antigen 4 from E. granulosus has an apparent molecular weight in excess of 400,000 daltons.

In conclusion, the data presented here indicate that parasite Pc carriers are lipoproteins and do not appear to be analogous to bacterial and fungal C-substances. In addition, it is clear that the proper- ties of the parasite lipoproteins are different from those of their mammal ian counterparts. Our con- tinued interest in these molecules is justified by observations from this and other laboratories which show that they are immunogenic infected hosts.

Further characterization of lipoproteins from A. suum is underway.

Acknowledgements--We thank Associate Professor Turner for a critical review of the manuscript and for providing A. lumbricoides and T. canis worms. We thank also Drs Dawkins and Fourie for the other parasite extracts used in this study, and Ms C. Jones for typing the manuscript. The financial assistance of the Princess Margaret Children's Medical Research Foundation is gratefully acknowledged. One of us (A.McW.) was the recipient of a Commonwealth Postgraduate Scholarship during the course of this work. This is publication No. 227 of the PMCMRF.

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