Veterinary Immunology and Immunopathology, 24 (1990) 11-25 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
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Characterization of Monoclonal Antibodies against Ovine Sarcocystis spp. Antigens by Immunoblotting and Immuno-electron Microscopy P. O'DONOGHUE 1, R. LUMB ~, P. SMITH 3, J. BROOKER 4 and N. MENCKE ~
1Central Veterinary Laboratories, Department of Agriculture, Adelaide, S.A. 5000 (Australia) 2Clinical Microbiology Division, and 3Tissue Pathology Division, Institute of Medical and Veterinary Science, Adelaide, S.A. 5000 (Australia) 4Waite Agricultural Research Institute, University of Adelaide, Adelaide, S.A. 5000 (Australia) ~Institut fi~r Parasitologie, Tieri~rztliche Hochschule, D-3000 Hannover 71 (Federal Republic of Germany) {Accepted 19 June 1989)
ABSTRACT O'Donoghue, P., Lumb, R., Smith, P., Brooker, J. and Mencke, N., 1990. Characterization of monoclonal antibodies against ovine Sarcocystis spp. antigens by immunoblottingand immunoelectron microscopy. Vet. Immunol. Immunopathol., 24: 11-25. Six monoclonal antibodies were raised in mice against purified cystozoite extracts of Sarcocystis gigantea and S. tenella from sheep. Each monoclonal antibody was evaluated for specificity by enzyme immunoassay, immunoblotting and immuno-electron microscopy using homologous and heterologous antigenic preparations. All six monoclonal antibodies exhibited good species-specifity when reacted against crude soluble cystozoite antigens in enzyme immunoassays. However, only two monoclonal antibodies (IgM and IgG2a) exhibited reactivity in Western blots against specific protein bands. Both reacted against S. gigantea antigens of 100 000, 43 000 and 39 000 molecular weight. Neither monoclonal antibody reacted against the heterologous species S. te neUa. Ultrastructural studies performed with colloidal-gold conjugated antisera revealed that both monoclonal antibodies reacted against antigens located around micronemes and amylopectin granules in S. gigantea cystozoites. Another monoclonal antibody (IgG0 reacted only against microneme determinants in S. tenella cystozoites. In contrast, polyclonal sheep and rabbit immune sera cross-reacted against a wide range of cystozoite antigens.
INTRODUCTION
Infections by the sporozoan parasites Sarcocystis spp. are of some concern to the sheep industry because certain species have been associated with carcass lesions and other species with animal mortality, morbidity and production losses. S. gigantea and S. medusiformis are transmitted to sheep by cats and 0165-2427/90/$03.50
© 1990 Elsevier Science Publishers B.V.
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P. 0'DONOGHUE ET AL.
both parasite species form visible (macroscopic) cysts in the skeletal muscles of sheep (Rommel et al., 1972; Collins et al., 1979). These macroscopic cysts have been classified under meat hygiene regulations as carcass lesions in several countries. In contrast, S. tenella and S. arieticanis are transmitted to sheep by dogs but both only form microscopic cysts in the musculature (Ford, 1974; Munday and Corbould, 1974; Heydorn, 1985). However, experimental infections performed with dog-borne species have been found to produce an acute and even lethal clinical disease (Munday et al., 1975), abortion (Leek and Fayer, 1978) and reductions in weight gain and wool growth (Munday, 1979; 1984). Infections are conventionally diagnosed post-mortem by the detection of muscle cysts during meat inspection or histopathological studies (Boch et al., 1979 ). The clinical diagnosis of dog-borne infections is also difficult due to the relatively nonspecific clinical signs of disease (Phillips, 1982). More recently, recourse has been made to the serodiagnosis of infections in livestock and although serum antibodies against Sarcocystis spp. can be demonstrated, no tests have been found to be species-specific due to the crude nature of the antigens employed and the marked cross-reactivity of polyclonal immune sera (Weiland et al., 1982; O'Donoghue and Weyreter, 1984). The present investigation was performed to raise monoclonal antibodies against individual Sarcocystis spp. from sheep and to determine their speciesspecificity by immunoblotting and immuno-electron microscopy. MATERIALSAND METHODS
Source o[parasites Parasite cystozoites were harvested according to techniques previously described by O'Donoghue et al. (1986). Briefly, S. teneUa cystozoites were harvested by trypsin digestion of the skeletal muscles of sheep 120 days after their experimental infection with 10 000 sporocysts from dogs. S. gigantea cystozoites were harvested from large visible cysts from the oesophageal muscles of naturally-infected sheep slaughtered at local metropolitan abattoirs. Purified cystozoites were stored frozen at - 80 ° C until use.
Polyacrylamide gel electrophoresis Cystozoite samples were subject to electrophoresis in 12% polyacrylamide gels using a high pH, discontinuous buffer system similar to that described by Lumb et al. (1988). Briefly, aliquots of solubilized cystozoites were loaded onto 4% polyacrylamide stacking gels cast over 12% polyacrylamide resolving gels (0.375 M Tris buffer pH 8.8, 0.1% SDS). The gels were subjected to electrophoresis at 25 mA (Bio-Rad Protean II Cell) until the bromophenol blue marker reached the bottom of the gels (4-5 h). The gels were stained with 0.2% Coom-
MONOCLONALANTIBODIESAGAINSTOVINESARCOCYSTISSPP.
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assie blue for 30 min and then destained for 2 days. High and low molecular weight (Mw) standards (Bio-Rad) were used to calibrate each gel thereby facilitating Mw determination. Polyclonal immune sera Serum samples were collected from 2 specific-pathogen-free (SPF) lambs 5 days before and 70 days after their experimental infection with 10 000 S. tenella sporocysts from dogs. Samples were also collected from another two SPF lambs at similar times after infection with 5000 S. gigantea sporocysts from cats. Antisera were also raised in rabbits immunized with soluble S. tenella or S. gigantea cystozoite preparations emulsified in Freund's complete adjuvant (FCA). The rabbits received a booster injection 35 days after immunization and were bled 5 days later. Monoclonal antibody production Hybridoma cell clones secreting monoclonal antibodies were produced according to the techniques described by Zola and Brooks (1982). BALB/c mice were immunized with soluble sonicate extracts of whole cystozoites of either S. gigantea or S. tenella emulsified with FCA. Additional mice were also immunized with specific cystozoite proteins harvested from polyacrylamide gels (Table 1 ). Horizontal strips containing discrete protein bands were cut from the gels, washed in several changes of Tris buffer (without additional SDS), thoroughly homogenized in phosphate-buffered saline (PB S ) pH 7.2 and then dialysed overnight against fresh PBS. Homogenated gel suspensions were then TABLE1 Antigenic preparations used to immunize mice for the production of monoclonal antibodies against Sarcocystis spp. from sheep Antigen
Monoclonal antibody
Parasite species
Antigen preparation
Mw range
Code
Isotype
S. gigantea
Sonicate extract from whole cystozoites Large Mw bands from polyacrylamide gels Small Mw bands from polyacrylamide gels Sonicate extract from whole cystozoites Small Mw bands from polyacrylamide gels Small Mw bands from polyacrylamide gels
10 000-200 000 (all bands) 65 000-100 000 (2 major bands ) 20 500-23 000 (2 major bands ) 10 000-200 000 ( all bands ) 48 000-65 500 (5 major bands ) 48 000-80 000 (8 major bands )
L3
IgG2a
G4
IgM
M3
IgM
$3
IgG2a
T2
IgG2~
T3
IgG1
S. gigantea S. gigantea S. teneUa S. tenella S. teneUa
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P. O'DONOGHUE ET AL.
emulsified in FCA and injected into mice. All mice received booster inoculations 28 days later by subcutaneous injection of similar inocula without FCA. Spleen cell suspensions were prepared from each of the immunized mice 4 days after booster injection and fused with myeloma cells (P3-x63-Ag8.653 cell line) by the addition of polyethylene glycol. The resultant hydridoma cells were cultured in hypoxanthine/aminopterin/thymidine selective medium and culture supernatants screened after 14 days for the presence of antibodies against S. gigantea and S. teneUa antigens by enzyme immunoassays. Positive cultures were then cloned by limiting dilution and supernatants tested 14 days later for the presence of specific antibodies by enzyme immunoassay.
Enzyme immunoassays Antibody titres against soluble cystozoite antigenic preparations of S. gigantea and S. teneUa were determined by enzyme-linked immunosorbent assays. The technique was similar to that described in detail by O'Donoghue and Weyreter (1984) except that the conjugate consisted of rabbit anti-mouse immunoglobulins labelled with horse-radish peroxidase (Nordic, Tilburg) and the substrate consisted of 42 m M tetramethylbenzidine in dimethylsulphoxide diluted 1/100 in 0.1M sodium acetate/citric acid buffer pH 6.0 containing 0.01% hydrogen peroxide. Reactions were stopped by the addition of 2M sulphuric acid and then read at a wavelength of 405 nm. Each monoclonal antibody was also characterized using a commercially-available isotyping kit (Misotest, Commonwealth Serum Laboratories). Immunoblots Protein bands were transferred from polyacrylamide gels to nitrocellulose paper according to the technique of Tsang et al. (1983). Briefly, polyacrylamide gels and similar-sized nitrocellulose filters (0.45 #m, Schleicher and Schuell, Dassel) were equilibrated in chilled Tris buffer containing 1.45% glycine and 20% methanol. Electrotransfer was conducted at 30 V overnight in a transblot cell (Bio-Rad) and the results checked by staining a small strip with 0.1% amido black. Nitrocellulose filters were washed in Tris-buffered saline pH 7.4 containing 0.05% Tween 20 (T-TBS) and then blocked using 3% bovine serum albumin (BSA). Individual filter paper strips were incubated with each monoclonal antibody diluted in T - T B S containing 3% BSA for 90 min at room temperature. The strips were washed three times in T-TBS and then further incubated with the conjugate (rabbit anti-mouse immunoglobulins labelled with horse-radish peroxidase) diluted in T - T B S containing 1% BSA. The filters were washed twice in T-TBS, once in TBS and then reacted in the dark for 110 min with the substrate consisting of 0.3% 4-chloro-l-naphthol dissolved in methanol and diluted immediately before use with four volumes of TBS containing 0.01% hydrogen peroxide. Reactions were stopped by placing the filters in distilled water.
MONOCLONAL ANTIBODIES AGAINST OVINE SARCOCYSTIS SPP.
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Immuno-electron microscopy Ultra-thin sections of cysts were incubated with each antibody preparation and an appropriate colloidal-gold conjugated antiserum according to the techniques described by Smith (1988). Briefly, sheep musculature containing mature S. gigantea or S. tenella cysts were fixed in 1% glutaraldehyde in PBS for 30 min and then washed three times in PBS. Small blocks were pre-treated with 1% a m m o n i u m chloride, washed in PBS, dehydrated in graded methanol solutions at - 2 5 °C and infiltrated with LR-White resin (London Resin Co., Surrey) for 2 h at - 2 5 °C and then 14 days at room temperature. The blocks were finally embedded in activated resin at - 25 ° C and slowly brought to room temperature once polymerized. Semi-thin sections (1/lm) stained with 0.1% toluidine blue were screened for cysts and ultra-thin sections (90 nm) were then cut and mounted on formvar-coated nickel grids. Sections were pre-incubated with 1% bovine serum albumin (BSA) in PBS for 10 min and then incubated with each of the monoclonal or polyclonal antibody preparations diluted 1/10 in PBS containing 1% BSA and 1% Tween 20 (BT-PBS) for 2h .The sections were washed three times in PBS, once in B T - P B S and then incubated for 30 min with the relevant anti-species immunoglobulins (goat anti-mouse, rabbit anti-sheep or goat anti-rabbit) conjugated to colloidal-gold (15 n m particles, E-Y Laboratories, San Mateo) and diluted 1/50 in BT-PBS. The sections were washed three times in PBS, twice in distilled water, stained with 2 % uranyl acetate and 0.1% bismuth subnitrate and then examined in a transmission electron microscope (Siemens Elmiskop, IA). Additional sections were also incubated with negative control mouse, sheep and rabbit sera to assess nonspecific background staining reactions. RESULTS
A total of 17 cloned hybridoma cell cultures were initially selected for detailed analysis on the basis of their apparent species-specificity in the screening enzyme immunoassays. However, only six cultures exhibited good growth characteristics, demonstrated persistence of antibody production and remained stable following cryopreservation. Immunoglobulins present in culture supernatants were characterized using a commercial test kit and only single heavy- and light-chain isotypes were detected in each preparation (Table 1 ). Each monoclonal antibody was then examined for antigen specificity by enzyme immunoassay, immunoblotting and immuno-electron microscopy.
Enzyme immunoassays The six monoclonal antibody preparations were reacted against crude soluble sonicate extracts of S. gigantea and S. tenella cystozoites. Each monoclonal antibody was found to be species-specific and only reacted against homologous antigen preparations (Fig. 1 ). Three monoclonal antibodies raised against S.
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P. O'DONOGHUE
ET AL.
T T
G
E=
2-
G
T
11
l-
Q;
1 / J
~T
,e_ 0
S.
M3
G4
L3
$3
T2
T3
Monoclonal antibody
Fig. 1. Reactivities of monoclonal antibodies raised against S. gigantea (L3, G4, M3) and S. teneUa ($3, T2, T3 ) in enzyme immunoassays using homologous and heterologous antigenic preparations; G = S. gigantea antigen, T = S. tenella antigen, C = negative serum control.
G l
T I
G i
I
T !
G i
T
!
I
+ +i++++++++:+i+i+ii+i+il
116.-~
+++++i-,~,,100
92 "~" 66,,-!~
45,-I~
31
21
14-]~
I
I
PAGE
.
I
I
BLOTS
Fig. 2. Electrophoretic profiles of S. gigantea (G) and S. teneUa (T) cystozoites in polyacrylamide gels {PAGE) and corresponding nitrocellulose immunoblots incubated with two monoclonal antibodies raised against S. gigantea (G4 and L3 ); numeric values represent molecular weights × 10~.
MONOCLONAL
ANTIBODIESAGAINSTOVINESARCOCYSTIS SPP.
]7
gigantea cystozoite extracts (L3, G4 and M3 ) reacted strongly against homologous S. gigantea antigenic preparations but not against heterologous S. teneUa preparations (as compared to background and negative control values). Con-
Fig. 3. Electron micrograph of S. gigantea cystozoites reacted with monoclonal antibody G4 demonstrating specific gold-particle labelling around micronemes and amylopectin granules. X 8000. Fig. 4. S. gigantea cystozoites reacted with monoclonal antibody L3 demonstrating specific labelling around micronemes and amylopectin granules. X 8000. Fig. 5. S. tenella cystozoite reacted with monoclonal antibody T3 exhibiting specific labelling of micronemes. X 8000. Fig. 6. S. teneUa cystozoite reacted with monoclonal antibody T3 exhibiting labelling of micronemes near conoid. X 8000.
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P. O'DONOGHUEET AL.
versely, three monoclonal antibodies raised against S. tenella ($3, T2 and T3 ) reacted strongly against homologous S. teneUa antigens but not against those of S. gigantea.
Electrophoretic characterization Soluble cystozoite extracts of each parasite species were subject to electrophoretic separation on polyacrylamide gels. The polypeptide banding profiles were found to be unique for each species examined (Fig. 2 ). A total of 55 bands were detected in S. gigantea extracts ranging in Mw from 12 000 to > 200 000 whereas 43 bands were detected in S. teneUa extracts in a similar Mw range. Although corresponding bands varied in their staining intensities, distinct differences were observed between the two parasite species. At least seven bands (28 500, 39 000, 43 000, 50 000, 57 000, 84 000 and 100 000 Mw) were present in S. gigantea samples but absent from S. tenella samples whereas at least nine bands (13 000, 14 500, 17 000, 22 500, 29 500, 40 500, 48 000, 71 000 and 87 000 Mw) were present only in S. teneUa samples. Other differences observed between the two species consisted mainly of groups of bands exhibiting unique patterns of intensity; e.g. very dense bands evident from 19 000 to 24 000 Mw for S. gigantea but from 14 000 to 18 000 Mw for S. tenella. Immunoblots Following electrophoresis, protein bands were transferred to nitrocellulose filters and then reacted with each monoclonal antibody. Positive reactions were only observed for two of the six monoclonal antibodies (G4 and L3). Both antibodies exhibited similar reactions against three discrete bands of S. giganFig. 7. Electron micrograph of S. gigantea cystozoites reacted with polyclonal rabbit antisera (raised against homologous antigen) demonstrating heavy labelling of micronemes. × 7000. Fig. 8. S. teneUa cyst reacted with homologous immune serum exhibiting labelling of primary cyst wall, rhoptries and cytoplasmic elements. × 8000. Fig. 9. S. teneUa cystozoite reacted with homologous immune serum demonstrating labelling of micronemes around conoid. × 7000. Fig. 10. S. gigantea cystozoite reacted with heterologous immune serum (anti-S. teneUa) exhibiting cross-reactive labelling of micronemes around conoid. × 7000. Fig. 11. S. gigantea cyst reacted with homologous immune serum demonstrating labelling of' primary cyst wall. × 8000. Fig. 12. S. gigantea cystozoites reacted with homologous immune serum exhibiting specific labelling of pellicle determinants. × 7000. Fig. 13. S. tenella cystozoite reacted with heterologous immune serum (anti-S. gigantea) demonstrating cross-reactive labelling of micronemes. × 7000.
MONOCLONAL ANTIBODIES AGAINST OVINE SARCOCYSTIS SPP.
19
tea only (Fig. 2). Both reacted strongly against one band of 100 000 Mw, slightly weaker against another band of 43 000 Mw and only very weakly against a third band of 39 000 Mw. Neither antibody reacted against any protein band of the heterologous species S. tenella. No reactivity was apparent in any immunoblot with the other four monoclonal antibodies (M3, $3, T2 or T3 ). Immuno-electron microscopy Ultra-thin sections of cysts were treated with each antibody preparation and specific reactions were visualized using colloidal-gold conjugates. No gold labelling was observed in any of the negative control preparations. Only three of
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P. O'DONOGHUE ET AL.
the six monoclonal antibodies exhibited specific labelling of cystozoite antigens. Two monoclonal antibodies (G4 and L3) demonstrated labelling of structures located around micronemes and occasionally around amylopectin granules in S. gigantea cystozoites (Figs. 3 and 4). These antibodies did not exhibit any apparent labelling of similar structures in cystozoites of the heterologous species S. tenella. Another monoclonal antibody (T3) also exhibited specific labelling around micronemes and sometimes around amylopectin granules but only in S. teneUa cystozoites (Figs. 5 and 6). No other organelles or membranes exhibited specific labelling with these or the other monoclonal antibodies. Polyclonal rabbit antisera raised against S. gigantea cystozoites demonstrated heavy labelling of micronemes in both S. gigantea and S. tenella cystozoites (e.g. Fig. 7). Similarly, antisera raised against S. teneUa cystozoites also demonstrated cross-reactive labelling of micronemes in cystozoites of both species. I m m u n e sera collected from experimentally-infected sheep exhibited marked cross-reactivity and labelled a variety of antigenic structures in cysts and cystozoites of both species. I m m u n e serum from sheep infected with S. tenella labelled the primary cyst wall surrounding the palisade-like protrusions of S. tenella cysts as well as rhoptries, micronemes and undefined cytoplasmic elements in cystozoites (Figs. 8 and 9). The same immune serum cross-reacted with similar elements in cystozoites of S. gigantea (Fig. 10). Immune serum from sheep infected with S. gigantea labelled the primary cyst wall surrounding the cauliflower-like protrusions of S. gigantea cysts (Fig. 11) as well as the cystozoite pellicle, micronemes and other undefined cytoplasmic elements (Fig. 12 ). This immune serum also cross-reacted with micronemes and cytoplasmic determinants in S. teneUa cystozoites (Fig. 13). DISCUSSION The results demonstrate the successful production of monoclonal antibodies against two Sarcocystis spp. from sheep and also reveal several limitations in the techniques used to characterize their antigenic specifity. Although six monoclonal antibodies were found to be species-specific in enzyme immunoassays, only two reacted against specific protein bands in immunoblots and only three reacted against discrete morphological determinants in immuno-electron microscopy studies. Mice were immunized with crude soluble cystozoite extracts of each parasitic species as well as with specific protein bands harvested from polyacrylamide gels. Although 17 cloned hybridomas were identified by the screening assays as producing antibodies of interest, only six were successfully maintained in the laboratory and continued to produce single immunoglobulin isotypes. Previous studies have shown that some hybridomas fail to thrive in culture, others lose their capacity to produce immunoglobulins, some are relatively fragile and
MONOCLONAL ANTIBODIES AGAINST OVINE SARCOCYSTIS SPP.
21
do not survive cryopreservation and others become overgrown with non-secreting lines (cf. Zola and Brooks, 1982). The six monoclonal antibodies all exhibited marked species-specificity in enzyme immunoassays when tested against homologous and heterologous antigenic preparations. Although monoclonal antibodies have previously been raised against S. muris cystozoites from mice (Entzeroth and Goerlich, 1987), they have not yet been tested against heterologous parasite species. In contrast, species-specific monoclonal antibodies have been produced against three different Eimeria spp. from chickens and another two species from turkeys (Danforth and Augustine, 1983 ). In order to characterize the antigenic specificities of the monoclonal antibodies, immunoblot studies were conducted against cystozoite proteins following their electrophoretic separation. Only two of the six monoclonal antibodies reacted against specific protein bands thereby indicating that the antigenic epitopes involved were conserved during sample preparation or were renatured in the nitrocellulose filters following detergent solubilization and electrophoretic transfer. The failure of four monoclonal antibodies to react in immunoblots suggests that their antigenic epitopes were destroyed, denatured or rendered inactive by the sample treatment. Similar negative results in immunoblotting studies have previously been reported for a variety of monoclonal antibodies and attributed to epitope denaturation (cf. Campbell, 1984). Alternative means of characterizing the antigenic specificities of these monoclonal antibodies can be employed which are less destructive in nature, such as gel fractionation or affinity chromatography. The two monoclonal antibodies which reacted against homologous S. gigantea antigens in immunoblots (L3 and G4) both recognized the same three antigens although they were produced by different vaccination regimes. Both recognized antigens of 100000, 43000 and 39000 Mw thereby suggesting that either three different proteins possessed the same antigen epitope or that the smaller Mw proteins were degradation products which retained a common epitope. Multiple bands previously recognized in other monoclonal antibody studies have been attributed to proteolytic degradation products, the presence of variable lipid or sugar moieties or to genuine epitope polymorphism (cf. Campbell, 1984). The three proteins recognized by the two monoclonal antibodies were also found to be unique to S. gigantea cystozoite extracts as demonstrated by polyacrylamide gel electrophoresis. Polyacrylamide electrophoretic studies have not previously been performed to compare polypeptide profiles of different Sarcocystis spp. from sheep. Two species from mice, S. muris and S. dispersa, have been compared in isoelectric focusing studies and found to have distinctive protein banding patterns (Reiter and Mareis, 1986). Several unique polypeptides were observed for S. gigantea and S. tenella from sheep although many common proteins varying only in intensity were found. The interpretation of eletrophoretic profiles relies largely on subjective assessments of banding patterns and can be markedly influenced
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P. O'DONOGHUE ET AL.
by differential sample treatments resulting in varying degrees of proteolysis or polypeptide degradation. Nonetheless, distinct qualitative differences (i.e. bands present or absent) were observed between S. tenella and S. gigantea which may be of some significance in future characterization studies. Various subcellular fractions ofS. gigantea cystozoites (erroneously named S. teneUa) have previously been analysed by polyacrylamide electrophoresis and numerous proteins have been characterized with respect to Mw from purified pellicular membranes, micronemes and dense granules (Dubremetz and Dissous, 1980). However, none of the proteins from these membranes or organelles correspond in Mw to the three S. gigantea proteins detected by the monoclonal antibodies in the immunoblots. Immuno-electron microscopic studies revealed that both monoclonal antibodies L3 and G4 recognized antigens located around micronemes and amylopectin granules within S. gigantea cystozoites. The exact sites of the antigens were difficult to determine but they appeared to be associated with membranes surrounding these organelles. Unfortunately, whilst routine post-fixation techniques do improve the resolution of ultrastructural features (particularly membranes), their use in immuno-labelling studies have been found to severely reduce antigen recognition by monoclonal and polyclonal antibodies (Ingram et al., 1988; Smith, 1988). Resolution is therefore often sacrificed to maintain antigenicity. Nonetheless, because the lipid contents of the amylopectin granules were removed during sample dehydration leaving only vacant spaces, the pattern of gold labelling around the periphery of these spaces suggested that the antigens were located on the surrounding membranes. Another monoclonal antibody (T3) exhibited specific labelling of antigens located around similar organelles but in cystozoites of the homologous species S. teneUa. Little antigenic cross-reactivity was evident between the different monoclonal antibodies indicating that different epitopes occured in similar locations in each parasite species. Monoclonal antibodies raised against other sporozoan parasites have been found to label rhoptry and microneme antigens in Plasmodium falciparum (Uni et al., 1987; Ingrain et al., 1988), merozoite surface antigens in P. knowlesi (Aikawa et al., 1986) and sporozoite surface antigens in E. tenella (Speer et al., 1983). To date, no studies have yet been published examining monoclonal antibody cross-reactivity between different genera of protozoa. The two polyclonal rabbit antisera raised against cystozoites of each parasite species were found to be markedly cross-reactive. S. gigantea and S. teneUa micronemes were heavily labelled by either antiserum. Because micronemes predominate as organelles in cystozoites, they would be expected to be major components of soluble extracts used for antiserum production. The high degree of cross-reactivity observed suggests that micronemes from different parasite species share many common antigen epitopes. In contrast, rabbit antisera raised
MONOCLONAL ANTIBODIES AGAINST OVINE SARCOCYSTIS SPP.
23
against S. muris and S. dispersa cystozoites were found to be relatively speciesspecific in immunoblot studies (Reiter and Mareis, 1986). Immune sera collected from sheep experimentally infected with S. gigantea or S. tenella were also found to be highly cross-reactive for a variety of cystozoite antigens; including pellicle membranes, micronemes, rhoptries and undefined cytoplasmic determinants. These elements must share common epitopes between species to account for the marked cross-reactivity. In addition, the primary cyst walls surrounding the cysts exhibited specific labelling in homologous test systems. Although these parasitic elements are located intracellularly within muscle fibres and should not be accessible to host antibodies, the fact that they are recognized by immune sera indicates that antigens must be released or exposed to the host immune system. Not all cysts are successful in their establishment, and degenerating cysts surrounded by mononuclear cellular infiltrates have previously been reported, occasionally in association with macrophage myophagia (cf. O'Donoghue and Wilkinson, 1988). A single report of active protein release or secretion has also been made involving the demonstration of exocytosis of dense granule contents of S. muris into the secondary parasitophorous vacuole (Entzeroth et al., 1986). In conclusion, the six monoclonal antibodies raised against S. gigantea and S. tenella antigens were found to be species-specific in enzyme immunoassays. The techniques of immunoblotting and immuno-electron microscopy were successful in further defining the antigenic specificities of several of the monoclonal antibodies. Nevertheless, their species-specificity remains to be confirmed by further testing against antigenic preparations of two other Sarcocystis spp. occuring in sheep, namely, S. medusiformis and S. arieticanis. If the monoclonal antibodies prove to be specific for individual parasite species, they would have great potential for use as diagnostic reagents or molecular probes. They could be incorporated directly into immunoassay or blot detection systems to detect parasite antigens in host fluids or they may be used in immunohistochemical studies to indentify parasites in biopsy or autopsy material. Alternatively, the monoclonal antibodies could be used to harvest specific antigens by affinity chromatography and the antigens then incorporated into immunoassays to detect host antibodies. Lastly, the monoclonal antibodies could be used as molecular probes in recombinant DNA studies to detect antigen expression following parasite gene cloning and insertion into suitable vectors. However, such potential uses are only speculative at this time and further detailed studies are required to determine their actual specificity and reactivity under various test conditions. ACKNOWLEDGEMENTS
The authors wish to thank Barbara Stokes, Kate Williams, Janet Stevenson, Jeanette Clarke and Ruth Davies for their expert technical assistance in the
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P. O'DONOGHUEET AL.
m a i n t e n a n c e o f h y b r i d o m a cell l i n e s , t h e p e r f o r m a n c e o f e n z y m e i m m u n o a s s a y s a n d p r e p a r i n g u l t r a - t h i n s e c t i o n s for e l e c t r o n m i c r o s c o p y .
REFERENCES Aikawa, M., David, P., Fine, E., Hudson, D., Klotz, F. and Miller, L.H., 1986. Localization of protective 143/140 kDa antigens of Plasmodium knowlesi by the use of antibodies and ultracryomicrotomy. Eur. J. Cell Biol., 41: 207-213. Boch, J., Bierschenck, A., Erber, M. and Weiland, G., 1979. Sarcocystis- und Toxoplasma-Infektionen bei Schlachtschafen in Bayern. Berl. Mtinch. Tier~irztl. Wochenschr., 92: 137-141. Campbell, A.M., 1984. Monoclonal antibody technology. The production and characterization of rodent and human hybridomas. In: R.H. Burdon and P.H. van Knippenberg (Editors), Laboratory Techniques in Biochemistry and Molecular Biology, vol. 13. Elsevier Science Publishers, Amsterdam, pp. 1-265. Collins, G.H., Atkinson, E. and Charleston, W.A.G., 1979. Studies on Sarcocystis species. III. The macrocystic species of sheep. N.Z. Vet. J., 27: 204-206. Danforth, H.D. and Augustine, P.C., 1983. Specificity and crossreactivity of immune serum and hybridoma antibodies to various species of avian coccidia. Poult. Sci., 62: 2145-2151. Dubremetz, J.F. and Dissous, C., 1980. Characteristic proteins of micronemes and dense granules from Sarcocystis tenella zoites (Protozoa, Coccidia), Mol. Biochem. Parasitol., 1: 279-289. Entzeroth, R. and Goerlich, R., 1987. Monoclonal antibodies against cystozoites of Sarcocystis muris (Protozoa, Apicomplexa). Parasitol. Res., 73: 568-570. Entzeroth, R., Dubremetz, J.F., Hodick, D. and Ferreira, E., 1986. Immunoelectron microscopic demonstration of the exocytosis of dense granule contents into the secondary parasitophorous vacuole of Sarcocystis muris (Protozoa, Apicomplexa). Eur. J. Cell Biol., 41: 182-188. Ford, G.E., 1974. Prey-predator transmission in the epizootiology of ovine Sarcosporidiosis. Aust. Vet. J., 50:38 39. Heydorn, A.O., 1985. Zur Entwicklung von Sarcocystis arieticanis n. sp. Berl. Mtinch. Tierfirztl. Wochenschr., 98: 231-241. Ingram, L.T., Stenzel, D.J., Kara, U.A.K. and Bushell, G.R, 1988. Localisation of internal antigens of Plasmodium [alciparum using monoclonal antibodies and colloidal gold. Parasitol. Res., 74: 208-215. Leek, R.G. and Fayer, R., 1978. Sheep experimentally infected with Sarcocystis from dogs. II. Abortion and disease in ewes. Cornell Vet., 68:108 123. Lumb, R., Lanser, J.A. and O'Donoghue, P.J., 1988. Electrophoretic and immunoblot analysis ol Crypto,sporidium oocysts. Immunol. Cell Biol., 66: 369-376. Munday, B.L., 1979. The effect of Sarcoeystis ovicanis on growth rate and haematocrit in lambs Vet. Parasitol., 5: 129-135. Munday, B.L., 1984. The effect ofSarcocystis tenella on wool growth in sheep. Vet. Parasitol., 15 91-94. Munday, B.L. and Corbould, A., 1974. The possible role of the dog in the epidemiology of ovin~ Sarcosporidiosis. Br. Vet. J., 130: 9-11. Munday, B.L., Barker, I.K. and Rickard, M.D., 1975. The developmental cycle of a species o Sarcocystis occurring in dogs and sheep, with observations on pathogenicity in the interme diate host. Z. Parasitenkd., 46:111 123. O'Donoghue, P.J. and Weyreter, H., 1984. Examinations on the serodiagnosis of Sarcocystis in fections. II Class-specific immunoglobulinresponses in mice, pigs and sheep. Zentralbl. Bak teriol. Hyg., I. Abt. Orig. A, 257: 168-184. O'Donoghue, P.J. and Wilkinson, R.G. 1988. Antibody development and cellular immune re
MONOCLONAL ANTIBODIES AGAINST OVINE SARCOCYSTIS SPP.
25
sponses in sheep immunized and challenged with Sarcocystis tenella sporocysts. Vet. Parasitol., 27: 251-265. O'Donoghue, P.J., Adams, M., Dixon, B.R., Ford, G.E. and Baverstock, P.R., 1986. Morphological and biochemical correlates in the characterization of Sarcocystis spp. J. Protozool., 33:114121. Phillips, P.H., 1982. The pathophysiology of Sarcocystis tenella infections in specific-pathogenfree (Sporozoa) sheep. Ph.D. Thesis, Adelaide University, 505 pp. Reiter, I. and Mareis, A., 1986. Zur Differenzierung von Sarcocystis muris- und S. dispersa-Infektionen der Maus mittels isoelektrischer Fokussierung und Immunoassays. Dtsch. Tier~rztl. Wochenschr., 93: 433-437. Rommel, M., Heydorn, A.O. and Gruber, F., 1972. Beitr~ige zum Lebenzyklus der Sarkosporidien. I. Die Sporozyste yon Sarcocystis teneUa in den Fiizes der Katze. Berl. Miinch. Tier~irztl. Wochenschr., 85: 101-105. Smith, P.S., 1988. Ultrastructural immuno-gold localization of immune deposits in human renal biopsies. Pathology, 20: 32-37. Speer, C.A., Wong, R.B. and Schenkel, R.H., 1983. Ultrastructural localization of monoclonal IgG antibodies for antigenic sites of Eirneria teneUa oocysts, sporocysts and sporozoites. J. Protozool., 30: 548-554. Tsang, V.C.W., Peralta, J.M. and Simons, A.R., 1983. Enzyme-linked immunoelectrotransfer blot techniques (EITB) for studying the specificities of antigens and antibodies separated by gel electrophoresis. Methods Enzymol., 92: 377-391. Uni, S., Masuda, A., Stewart, M.J., Igarashi, I., Nussenzweig, R. and Aikawa, M., 1987. Ultrastructural localization of the 150/130 Kd antigens in sexual and asexual blood stages of Plasrnodium falciparum-infected human erythrocytes. Am. J. Trop. Med. Hyg., 36: 481-488. Weiland, G., Reiter, I. and Boch, J., 1982. MSglichkeiten und Grenzen des serologischen Nachweises von Sarkosporidieninfektionen. Berl. Mtinch. Tier~irztl. Wochenschr., 95: 387-392. Zola, H. and Brooks, D., 1982. Techniques for the production and characterization of monoclonal hybridoma antibodies. In: J.G.R. Hurrell (Editor), Monoclonal Hybridoma Antibodies: Techniques and Application. C.R.C. Press, FL, pp. 1-57.
11
Characterization of Monoclonal Antibodies against Ovine Sarcocystis spp. Antigens by Immunoblotting and Immuno-electron Microscopy P. O'DONOGHUE 1, R. LUMB ~, P. SMITH 3, J. BROOKER 4 and N. MENCKE ~
1Central Veterinary Laboratories, Department of Agriculture, Adelaide, S.A. 5000 (Australia) 2Clinical Microbiology Division, and 3Tissue Pathology Division, Institute of Medical and Veterinary Science, Adelaide, S.A. 5000 (Australia) 4Waite Agricultural Research Institute, University of Adelaide, Adelaide, S.A. 5000 (Australia) ~Institut fi~r Parasitologie, Tieri~rztliche Hochschule, D-3000 Hannover 71 (Federal Republic of Germany) {Accepted 19 June 1989)
ABSTRACT O'Donoghue, P., Lumb, R., Smith, P., Brooker, J. and Mencke, N., 1990. Characterization of monoclonal antibodies against ovine Sarcocystis spp. antigens by immunoblottingand immunoelectron microscopy. Vet. Immunol. Immunopathol., 24: 11-25. Six monoclonal antibodies were raised in mice against purified cystozoite extracts of Sarcocystis gigantea and S. tenella from sheep. Each monoclonal antibody was evaluated for specificity by enzyme immunoassay, immunoblotting and immuno-electron microscopy using homologous and heterologous antigenic preparations. All six monoclonal antibodies exhibited good species-specifity when reacted against crude soluble cystozoite antigens in enzyme immunoassays. However, only two monoclonal antibodies (IgM and IgG2a) exhibited reactivity in Western blots against specific protein bands. Both reacted against S. gigantea antigens of 100 000, 43 000 and 39 000 molecular weight. Neither monoclonal antibody reacted against the heterologous species S. te neUa. Ultrastructural studies performed with colloidal-gold conjugated antisera revealed that both monoclonal antibodies reacted against antigens located around micronemes and amylopectin granules in S. gigantea cystozoites. Another monoclonal antibody (IgG0 reacted only against microneme determinants in S. tenella cystozoites. In contrast, polyclonal sheep and rabbit immune sera cross-reacted against a wide range of cystozoite antigens.
INTRODUCTION
Infections by the sporozoan parasites Sarcocystis spp. are of some concern to the sheep industry because certain species have been associated with carcass lesions and other species with animal mortality, morbidity and production losses. S. gigantea and S. medusiformis are transmitted to sheep by cats and 0165-2427/90/$03.50
© 1990 Elsevier Science Publishers B.V.
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P. 0'DONOGHUE ET AL.
both parasite species form visible (macroscopic) cysts in the skeletal muscles of sheep (Rommel et al., 1972; Collins et al., 1979). These macroscopic cysts have been classified under meat hygiene regulations as carcass lesions in several countries. In contrast, S. tenella and S. arieticanis are transmitted to sheep by dogs but both only form microscopic cysts in the musculature (Ford, 1974; Munday and Corbould, 1974; Heydorn, 1985). However, experimental infections performed with dog-borne species have been found to produce an acute and even lethal clinical disease (Munday et al., 1975), abortion (Leek and Fayer, 1978) and reductions in weight gain and wool growth (Munday, 1979; 1984). Infections are conventionally diagnosed post-mortem by the detection of muscle cysts during meat inspection or histopathological studies (Boch et al., 1979 ). The clinical diagnosis of dog-borne infections is also difficult due to the relatively nonspecific clinical signs of disease (Phillips, 1982). More recently, recourse has been made to the serodiagnosis of infections in livestock and although serum antibodies against Sarcocystis spp. can be demonstrated, no tests have been found to be species-specific due to the crude nature of the antigens employed and the marked cross-reactivity of polyclonal immune sera (Weiland et al., 1982; O'Donoghue and Weyreter, 1984). The present investigation was performed to raise monoclonal antibodies against individual Sarcocystis spp. from sheep and to determine their speciesspecificity by immunoblotting and immuno-electron microscopy. MATERIALSAND METHODS
Source o[parasites Parasite cystozoites were harvested according to techniques previously described by O'Donoghue et al. (1986). Briefly, S. teneUa cystozoites were harvested by trypsin digestion of the skeletal muscles of sheep 120 days after their experimental infection with 10 000 sporocysts from dogs. S. gigantea cystozoites were harvested from large visible cysts from the oesophageal muscles of naturally-infected sheep slaughtered at local metropolitan abattoirs. Purified cystozoites were stored frozen at - 80 ° C until use.
Polyacrylamide gel electrophoresis Cystozoite samples were subject to electrophoresis in 12% polyacrylamide gels using a high pH, discontinuous buffer system similar to that described by Lumb et al. (1988). Briefly, aliquots of solubilized cystozoites were loaded onto 4% polyacrylamide stacking gels cast over 12% polyacrylamide resolving gels (0.375 M Tris buffer pH 8.8, 0.1% SDS). The gels were subjected to electrophoresis at 25 mA (Bio-Rad Protean II Cell) until the bromophenol blue marker reached the bottom of the gels (4-5 h). The gels were stained with 0.2% Coom-
MONOCLONALANTIBODIESAGAINSTOVINESARCOCYSTISSPP.
13
assie blue for 30 min and then destained for 2 days. High and low molecular weight (Mw) standards (Bio-Rad) were used to calibrate each gel thereby facilitating Mw determination. Polyclonal immune sera Serum samples were collected from 2 specific-pathogen-free (SPF) lambs 5 days before and 70 days after their experimental infection with 10 000 S. tenella sporocysts from dogs. Samples were also collected from another two SPF lambs at similar times after infection with 5000 S. gigantea sporocysts from cats. Antisera were also raised in rabbits immunized with soluble S. tenella or S. gigantea cystozoite preparations emulsified in Freund's complete adjuvant (FCA). The rabbits received a booster injection 35 days after immunization and were bled 5 days later. Monoclonal antibody production Hybridoma cell clones secreting monoclonal antibodies were produced according to the techniques described by Zola and Brooks (1982). BALB/c mice were immunized with soluble sonicate extracts of whole cystozoites of either S. gigantea or S. tenella emulsified with FCA. Additional mice were also immunized with specific cystozoite proteins harvested from polyacrylamide gels (Table 1 ). Horizontal strips containing discrete protein bands were cut from the gels, washed in several changes of Tris buffer (without additional SDS), thoroughly homogenized in phosphate-buffered saline (PB S ) pH 7.2 and then dialysed overnight against fresh PBS. Homogenated gel suspensions were then TABLE1 Antigenic preparations used to immunize mice for the production of monoclonal antibodies against Sarcocystis spp. from sheep Antigen
Monoclonal antibody
Parasite species
Antigen preparation
Mw range
Code
Isotype
S. gigantea
Sonicate extract from whole cystozoites Large Mw bands from polyacrylamide gels Small Mw bands from polyacrylamide gels Sonicate extract from whole cystozoites Small Mw bands from polyacrylamide gels Small Mw bands from polyacrylamide gels
10 000-200 000 (all bands) 65 000-100 000 (2 major bands ) 20 500-23 000 (2 major bands ) 10 000-200 000 ( all bands ) 48 000-65 500 (5 major bands ) 48 000-80 000 (8 major bands )
L3
IgG2a
G4
IgM
M3
IgM
$3
IgG2a
T2
IgG2~
T3
IgG1
S. gigantea S. gigantea S. teneUa S. tenella S. teneUa
14
P. O'DONOGHUE ET AL.
emulsified in FCA and injected into mice. All mice received booster inoculations 28 days later by subcutaneous injection of similar inocula without FCA. Spleen cell suspensions were prepared from each of the immunized mice 4 days after booster injection and fused with myeloma cells (P3-x63-Ag8.653 cell line) by the addition of polyethylene glycol. The resultant hydridoma cells were cultured in hypoxanthine/aminopterin/thymidine selective medium and culture supernatants screened after 14 days for the presence of antibodies against S. gigantea and S. teneUa antigens by enzyme immunoassays. Positive cultures were then cloned by limiting dilution and supernatants tested 14 days later for the presence of specific antibodies by enzyme immunoassay.
Enzyme immunoassays Antibody titres against soluble cystozoite antigenic preparations of S. gigantea and S. teneUa were determined by enzyme-linked immunosorbent assays. The technique was similar to that described in detail by O'Donoghue and Weyreter (1984) except that the conjugate consisted of rabbit anti-mouse immunoglobulins labelled with horse-radish peroxidase (Nordic, Tilburg) and the substrate consisted of 42 m M tetramethylbenzidine in dimethylsulphoxide diluted 1/100 in 0.1M sodium acetate/citric acid buffer pH 6.0 containing 0.01% hydrogen peroxide. Reactions were stopped by the addition of 2M sulphuric acid and then read at a wavelength of 405 nm. Each monoclonal antibody was also characterized using a commercially-available isotyping kit (Misotest, Commonwealth Serum Laboratories). Immunoblots Protein bands were transferred from polyacrylamide gels to nitrocellulose paper according to the technique of Tsang et al. (1983). Briefly, polyacrylamide gels and similar-sized nitrocellulose filters (0.45 #m, Schleicher and Schuell, Dassel) were equilibrated in chilled Tris buffer containing 1.45% glycine and 20% methanol. Electrotransfer was conducted at 30 V overnight in a transblot cell (Bio-Rad) and the results checked by staining a small strip with 0.1% amido black. Nitrocellulose filters were washed in Tris-buffered saline pH 7.4 containing 0.05% Tween 20 (T-TBS) and then blocked using 3% bovine serum albumin (BSA). Individual filter paper strips were incubated with each monoclonal antibody diluted in T - T B S containing 3% BSA for 90 min at room temperature. The strips were washed three times in T-TBS and then further incubated with the conjugate (rabbit anti-mouse immunoglobulins labelled with horse-radish peroxidase) diluted in T - T B S containing 1% BSA. The filters were washed twice in T-TBS, once in TBS and then reacted in the dark for 110 min with the substrate consisting of 0.3% 4-chloro-l-naphthol dissolved in methanol and diluted immediately before use with four volumes of TBS containing 0.01% hydrogen peroxide. Reactions were stopped by placing the filters in distilled water.
MONOCLONAL ANTIBODIES AGAINST OVINE SARCOCYSTIS SPP.
15
Immuno-electron microscopy Ultra-thin sections of cysts were incubated with each antibody preparation and an appropriate colloidal-gold conjugated antiserum according to the techniques described by Smith (1988). Briefly, sheep musculature containing mature S. gigantea or S. tenella cysts were fixed in 1% glutaraldehyde in PBS for 30 min and then washed three times in PBS. Small blocks were pre-treated with 1% a m m o n i u m chloride, washed in PBS, dehydrated in graded methanol solutions at - 2 5 °C and infiltrated with LR-White resin (London Resin Co., Surrey) for 2 h at - 2 5 °C and then 14 days at room temperature. The blocks were finally embedded in activated resin at - 25 ° C and slowly brought to room temperature once polymerized. Semi-thin sections (1/lm) stained with 0.1% toluidine blue were screened for cysts and ultra-thin sections (90 nm) were then cut and mounted on formvar-coated nickel grids. Sections were pre-incubated with 1% bovine serum albumin (BSA) in PBS for 10 min and then incubated with each of the monoclonal or polyclonal antibody preparations diluted 1/10 in PBS containing 1% BSA and 1% Tween 20 (BT-PBS) for 2h .The sections were washed three times in PBS, once in B T - P B S and then incubated for 30 min with the relevant anti-species immunoglobulins (goat anti-mouse, rabbit anti-sheep or goat anti-rabbit) conjugated to colloidal-gold (15 n m particles, E-Y Laboratories, San Mateo) and diluted 1/50 in BT-PBS. The sections were washed three times in PBS, twice in distilled water, stained with 2 % uranyl acetate and 0.1% bismuth subnitrate and then examined in a transmission electron microscope (Siemens Elmiskop, IA). Additional sections were also incubated with negative control mouse, sheep and rabbit sera to assess nonspecific background staining reactions. RESULTS
A total of 17 cloned hybridoma cell cultures were initially selected for detailed analysis on the basis of their apparent species-specificity in the screening enzyme immunoassays. However, only six cultures exhibited good growth characteristics, demonstrated persistence of antibody production and remained stable following cryopreservation. Immunoglobulins present in culture supernatants were characterized using a commercial test kit and only single heavy- and light-chain isotypes were detected in each preparation (Table 1 ). Each monoclonal antibody was then examined for antigen specificity by enzyme immunoassay, immunoblotting and immuno-electron microscopy.
Enzyme immunoassays The six monoclonal antibody preparations were reacted against crude soluble sonicate extracts of S. gigantea and S. tenella cystozoites. Each monoclonal antibody was found to be species-specific and only reacted against homologous antigen preparations (Fig. 1 ). Three monoclonal antibodies raised against S.
16
P. O'DONOGHUE
ET AL.
T T
G
E=
2-
G
T
11
l-
Q;
1 / J
~T
,e_ 0
S.
M3
G4
L3
$3
T2
T3
Monoclonal antibody
Fig. 1. Reactivities of monoclonal antibodies raised against S. gigantea (L3, G4, M3) and S. teneUa ($3, T2, T3 ) in enzyme immunoassays using homologous and heterologous antigenic preparations; G = S. gigantea antigen, T = S. tenella antigen, C = negative serum control.
G l
T I
G i
I
T !
G i
T
!
I
+ +i++++++++:+i+i+ii+i+il
116.-~
+++++i-,~,,100
92 "~" 66,,-!~
45,-I~
31
21
14-]~
I
I
PAGE
.
I
I
BLOTS
Fig. 2. Electrophoretic profiles of S. gigantea (G) and S. teneUa (T) cystozoites in polyacrylamide gels {PAGE) and corresponding nitrocellulose immunoblots incubated with two monoclonal antibodies raised against S. gigantea (G4 and L3 ); numeric values represent molecular weights × 10~.
MONOCLONAL
ANTIBODIESAGAINSTOVINESARCOCYSTIS SPP.
]7
gigantea cystozoite extracts (L3, G4 and M3 ) reacted strongly against homologous S. gigantea antigenic preparations but not against heterologous S. teneUa preparations (as compared to background and negative control values). Con-
Fig. 3. Electron micrograph of S. gigantea cystozoites reacted with monoclonal antibody G4 demonstrating specific gold-particle labelling around micronemes and amylopectin granules. X 8000. Fig. 4. S. gigantea cystozoites reacted with monoclonal antibody L3 demonstrating specific labelling around micronemes and amylopectin granules. X 8000. Fig. 5. S. tenella cystozoite reacted with monoclonal antibody T3 exhibiting specific labelling of micronemes. X 8000. Fig. 6. S. teneUa cystozoite reacted with monoclonal antibody T3 exhibiting labelling of micronemes near conoid. X 8000.
18
P. O'DONOGHUEET AL.
versely, three monoclonal antibodies raised against S. tenella ($3, T2 and T3 ) reacted strongly against homologous S. teneUa antigens but not against those of S. gigantea.
Electrophoretic characterization Soluble cystozoite extracts of each parasite species were subject to electrophoretic separation on polyacrylamide gels. The polypeptide banding profiles were found to be unique for each species examined (Fig. 2 ). A total of 55 bands were detected in S. gigantea extracts ranging in Mw from 12 000 to > 200 000 whereas 43 bands were detected in S. teneUa extracts in a similar Mw range. Although corresponding bands varied in their staining intensities, distinct differences were observed between the two parasite species. At least seven bands (28 500, 39 000, 43 000, 50 000, 57 000, 84 000 and 100 000 Mw) were present in S. gigantea samples but absent from S. tenella samples whereas at least nine bands (13 000, 14 500, 17 000, 22 500, 29 500, 40 500, 48 000, 71 000 and 87 000 Mw) were present only in S. teneUa samples. Other differences observed between the two species consisted mainly of groups of bands exhibiting unique patterns of intensity; e.g. very dense bands evident from 19 000 to 24 000 Mw for S. gigantea but from 14 000 to 18 000 Mw for S. tenella. Immunoblots Following electrophoresis, protein bands were transferred to nitrocellulose filters and then reacted with each monoclonal antibody. Positive reactions were only observed for two of the six monoclonal antibodies (G4 and L3). Both antibodies exhibited similar reactions against three discrete bands of S. giganFig. 7. Electron micrograph of S. gigantea cystozoites reacted with polyclonal rabbit antisera (raised against homologous antigen) demonstrating heavy labelling of micronemes. × 7000. Fig. 8. S. teneUa cyst reacted with homologous immune serum exhibiting labelling of primary cyst wall, rhoptries and cytoplasmic elements. × 8000. Fig. 9. S. teneUa cystozoite reacted with homologous immune serum demonstrating labelling of micronemes around conoid. × 7000. Fig. 10. S. gigantea cystozoite reacted with heterologous immune serum (anti-S. teneUa) exhibiting cross-reactive labelling of micronemes around conoid. × 7000. Fig. 11. S. gigantea cyst reacted with homologous immune serum demonstrating labelling of' primary cyst wall. × 8000. Fig. 12. S. gigantea cystozoites reacted with homologous immune serum exhibiting specific labelling of pellicle determinants. × 7000. Fig. 13. S. tenella cystozoite reacted with heterologous immune serum (anti-S. gigantea) demonstrating cross-reactive labelling of micronemes. × 7000.
MONOCLONAL ANTIBODIES AGAINST OVINE SARCOCYSTIS SPP.
19
tea only (Fig. 2). Both reacted strongly against one band of 100 000 Mw, slightly weaker against another band of 43 000 Mw and only very weakly against a third band of 39 000 Mw. Neither antibody reacted against any protein band of the heterologous species S. tenella. No reactivity was apparent in any immunoblot with the other four monoclonal antibodies (M3, $3, T2 or T3 ). Immuno-electron microscopy Ultra-thin sections of cysts were treated with each antibody preparation and specific reactions were visualized using colloidal-gold conjugates. No gold labelling was observed in any of the negative control preparations. Only three of
20
P. O'DONOGHUE ET AL.
the six monoclonal antibodies exhibited specific labelling of cystozoite antigens. Two monoclonal antibodies (G4 and L3) demonstrated labelling of structures located around micronemes and occasionally around amylopectin granules in S. gigantea cystozoites (Figs. 3 and 4). These antibodies did not exhibit any apparent labelling of similar structures in cystozoites of the heterologous species S. tenella. Another monoclonal antibody (T3) also exhibited specific labelling around micronemes and sometimes around amylopectin granules but only in S. teneUa cystozoites (Figs. 5 and 6). No other organelles or membranes exhibited specific labelling with these or the other monoclonal antibodies. Polyclonal rabbit antisera raised against S. gigantea cystozoites demonstrated heavy labelling of micronemes in both S. gigantea and S. tenella cystozoites (e.g. Fig. 7). Similarly, antisera raised against S. teneUa cystozoites also demonstrated cross-reactive labelling of micronemes in cystozoites of both species. I m m u n e sera collected from experimentally-infected sheep exhibited marked cross-reactivity and labelled a variety of antigenic structures in cysts and cystozoites of both species. I m m u n e serum from sheep infected with S. tenella labelled the primary cyst wall surrounding the palisade-like protrusions of S. tenella cysts as well as rhoptries, micronemes and undefined cytoplasmic elements in cystozoites (Figs. 8 and 9). The same immune serum cross-reacted with similar elements in cystozoites of S. gigantea (Fig. 10). Immune serum from sheep infected with S. gigantea labelled the primary cyst wall surrounding the cauliflower-like protrusions of S. gigantea cysts (Fig. 11) as well as the cystozoite pellicle, micronemes and other undefined cytoplasmic elements (Fig. 12 ). This immune serum also cross-reacted with micronemes and cytoplasmic determinants in S. teneUa cystozoites (Fig. 13). DISCUSSION The results demonstrate the successful production of monoclonal antibodies against two Sarcocystis spp. from sheep and also reveal several limitations in the techniques used to characterize their antigenic specifity. Although six monoclonal antibodies were found to be species-specific in enzyme immunoassays, only two reacted against specific protein bands in immunoblots and only three reacted against discrete morphological determinants in immuno-electron microscopy studies. Mice were immunized with crude soluble cystozoite extracts of each parasitic species as well as with specific protein bands harvested from polyacrylamide gels. Although 17 cloned hybridomas were identified by the screening assays as producing antibodies of interest, only six were successfully maintained in the laboratory and continued to produce single immunoglobulin isotypes. Previous studies have shown that some hybridomas fail to thrive in culture, others lose their capacity to produce immunoglobulins, some are relatively fragile and
MONOCLONAL ANTIBODIES AGAINST OVINE SARCOCYSTIS SPP.
21
do not survive cryopreservation and others become overgrown with non-secreting lines (cf. Zola and Brooks, 1982). The six monoclonal antibodies all exhibited marked species-specificity in enzyme immunoassays when tested against homologous and heterologous antigenic preparations. Although monoclonal antibodies have previously been raised against S. muris cystozoites from mice (Entzeroth and Goerlich, 1987), they have not yet been tested against heterologous parasite species. In contrast, species-specific monoclonal antibodies have been produced against three different Eimeria spp. from chickens and another two species from turkeys (Danforth and Augustine, 1983 ). In order to characterize the antigenic specificities of the monoclonal antibodies, immunoblot studies were conducted against cystozoite proteins following their electrophoretic separation. Only two of the six monoclonal antibodies reacted against specific protein bands thereby indicating that the antigenic epitopes involved were conserved during sample preparation or were renatured in the nitrocellulose filters following detergent solubilization and electrophoretic transfer. The failure of four monoclonal antibodies to react in immunoblots suggests that their antigenic epitopes were destroyed, denatured or rendered inactive by the sample treatment. Similar negative results in immunoblotting studies have previously been reported for a variety of monoclonal antibodies and attributed to epitope denaturation (cf. Campbell, 1984). Alternative means of characterizing the antigenic specificities of these monoclonal antibodies can be employed which are less destructive in nature, such as gel fractionation or affinity chromatography. The two monoclonal antibodies which reacted against homologous S. gigantea antigens in immunoblots (L3 and G4) both recognized the same three antigens although they were produced by different vaccination regimes. Both recognized antigens of 100000, 43000 and 39000 Mw thereby suggesting that either three different proteins possessed the same antigen epitope or that the smaller Mw proteins were degradation products which retained a common epitope. Multiple bands previously recognized in other monoclonal antibody studies have been attributed to proteolytic degradation products, the presence of variable lipid or sugar moieties or to genuine epitope polymorphism (cf. Campbell, 1984). The three proteins recognized by the two monoclonal antibodies were also found to be unique to S. gigantea cystozoite extracts as demonstrated by polyacrylamide gel electrophoresis. Polyacrylamide electrophoretic studies have not previously been performed to compare polypeptide profiles of different Sarcocystis spp. from sheep. Two species from mice, S. muris and S. dispersa, have been compared in isoelectric focusing studies and found to have distinctive protein banding patterns (Reiter and Mareis, 1986). Several unique polypeptides were observed for S. gigantea and S. tenella from sheep although many common proteins varying only in intensity were found. The interpretation of eletrophoretic profiles relies largely on subjective assessments of banding patterns and can be markedly influenced
22
P. O'DONOGHUE ET AL.
by differential sample treatments resulting in varying degrees of proteolysis or polypeptide degradation. Nonetheless, distinct qualitative differences (i.e. bands present or absent) were observed between S. tenella and S. gigantea which may be of some significance in future characterization studies. Various subcellular fractions ofS. gigantea cystozoites (erroneously named S. teneUa) have previously been analysed by polyacrylamide electrophoresis and numerous proteins have been characterized with respect to Mw from purified pellicular membranes, micronemes and dense granules (Dubremetz and Dissous, 1980). However, none of the proteins from these membranes or organelles correspond in Mw to the three S. gigantea proteins detected by the monoclonal antibodies in the immunoblots. Immuno-electron microscopic studies revealed that both monoclonal antibodies L3 and G4 recognized antigens located around micronemes and amylopectin granules within S. gigantea cystozoites. The exact sites of the antigens were difficult to determine but they appeared to be associated with membranes surrounding these organelles. Unfortunately, whilst routine post-fixation techniques do improve the resolution of ultrastructural features (particularly membranes), their use in immuno-labelling studies have been found to severely reduce antigen recognition by monoclonal and polyclonal antibodies (Ingram et al., 1988; Smith, 1988). Resolution is therefore often sacrificed to maintain antigenicity. Nonetheless, because the lipid contents of the amylopectin granules were removed during sample dehydration leaving only vacant spaces, the pattern of gold labelling around the periphery of these spaces suggested that the antigens were located on the surrounding membranes. Another monoclonal antibody (T3) exhibited specific labelling of antigens located around similar organelles but in cystozoites of the homologous species S. teneUa. Little antigenic cross-reactivity was evident between the different monoclonal antibodies indicating that different epitopes occured in similar locations in each parasite species. Monoclonal antibodies raised against other sporozoan parasites have been found to label rhoptry and microneme antigens in Plasmodium falciparum (Uni et al., 1987; Ingrain et al., 1988), merozoite surface antigens in P. knowlesi (Aikawa et al., 1986) and sporozoite surface antigens in E. tenella (Speer et al., 1983). To date, no studies have yet been published examining monoclonal antibody cross-reactivity between different genera of protozoa. The two polyclonal rabbit antisera raised against cystozoites of each parasite species were found to be markedly cross-reactive. S. gigantea and S. teneUa micronemes were heavily labelled by either antiserum. Because micronemes predominate as organelles in cystozoites, they would be expected to be major components of soluble extracts used for antiserum production. The high degree of cross-reactivity observed suggests that micronemes from different parasite species share many common antigen epitopes. In contrast, rabbit antisera raised
MONOCLONAL ANTIBODIES AGAINST OVINE SARCOCYSTIS SPP.
23
against S. muris and S. dispersa cystozoites were found to be relatively speciesspecific in immunoblot studies (Reiter and Mareis, 1986). Immune sera collected from sheep experimentally infected with S. gigantea or S. tenella were also found to be highly cross-reactive for a variety of cystozoite antigens; including pellicle membranes, micronemes, rhoptries and undefined cytoplasmic determinants. These elements must share common epitopes between species to account for the marked cross-reactivity. In addition, the primary cyst walls surrounding the cysts exhibited specific labelling in homologous test systems. Although these parasitic elements are located intracellularly within muscle fibres and should not be accessible to host antibodies, the fact that they are recognized by immune sera indicates that antigens must be released or exposed to the host immune system. Not all cysts are successful in their establishment, and degenerating cysts surrounded by mononuclear cellular infiltrates have previously been reported, occasionally in association with macrophage myophagia (cf. O'Donoghue and Wilkinson, 1988). A single report of active protein release or secretion has also been made involving the demonstration of exocytosis of dense granule contents of S. muris into the secondary parasitophorous vacuole (Entzeroth et al., 1986). In conclusion, the six monoclonal antibodies raised against S. gigantea and S. tenella antigens were found to be species-specific in enzyme immunoassays. The techniques of immunoblotting and immuno-electron microscopy were successful in further defining the antigenic specificities of several of the monoclonal antibodies. Nevertheless, their species-specificity remains to be confirmed by further testing against antigenic preparations of two other Sarcocystis spp. occuring in sheep, namely, S. medusiformis and S. arieticanis. If the monoclonal antibodies prove to be specific for individual parasite species, they would have great potential for use as diagnostic reagents or molecular probes. They could be incorporated directly into immunoassay or blot detection systems to detect parasite antigens in host fluids or they may be used in immunohistochemical studies to indentify parasites in biopsy or autopsy material. Alternatively, the monoclonal antibodies could be used to harvest specific antigens by affinity chromatography and the antigens then incorporated into immunoassays to detect host antibodies. Lastly, the monoclonal antibodies could be used as molecular probes in recombinant DNA studies to detect antigen expression following parasite gene cloning and insertion into suitable vectors. However, such potential uses are only speculative at this time and further detailed studies are required to determine their actual specificity and reactivity under various test conditions. ACKNOWLEDGEMENTS
The authors wish to thank Barbara Stokes, Kate Williams, Janet Stevenson, Jeanette Clarke and Ruth Davies for their expert technical assistance in the
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P. O'DONOGHUEET AL.
m a i n t e n a n c e o f h y b r i d o m a cell l i n e s , t h e p e r f o r m a n c e o f e n z y m e i m m u n o a s s a y s a n d p r e p a r i n g u l t r a - t h i n s e c t i o n s for e l e c t r o n m i c r o s c o p y .
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