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Theileria parva: Expression of a sporozoite surface coat antigen
EXPERIMENTAL PARASITOLOGY60, 90--100 (1985)
Theileria parva: Expression of a Sporozoite Surface Coat Antigen DIRK A. E. DOBBELAERE, 1 PAUL WEBSTER, BRIAN L. LEITCH, 2 W O L F P. VOIGT, AND A N T H O N Y D . IRVIN International Laboratory for Research on Animal Diseases, P.O. Box 30709, Nairobi, Kenya (Accepted for publication 11 December 1984) DOBBELAERE, D. A. E., WEBSTER, P., LEITCH, B. L., VOIGT, W. P., AND IRVIN, A. D. 1985. Theileria parva: Expression of a sporozoite surface coat antigen. Experimental Parasitology 60: 90-100. A monoclonal antibody specific for the Theileria parva sporozoite, which recognizes a determinant on the surface coat and blocks sporozoite infectivity, was used to investigate the presence of the determinant on other stages of the parasite lifecycle. Immunofluorescence techniques did not demonstrate this determinant on the kinete, schizont, merozoite, or piroplasm stages of the parasite. Immunoautoradiography, using a tritiated form of the monoclonal antibody, on sections of infected salivary glands collected from ticks that had fed for 0, 1, 2, 3, or 4 days revealed that the determinant recognized was synthesized predominantly during sporogony, between 2 to 3 days after the tick started feeding. Immunoelectron microscopy was performed on ultrathin frozen sections of infected tick salivary glands incubated with the monoclonal antibody followed by Protein-A-colloidal gold. The antigen or its precursor could be detected in the developing parasite. In ticks fed 2 days, the sporoblast was labeled, both in the cytoplasm and on parasite membranes, often including the nuclear envelope. In sections from ticks fed 4 days, the sporozoite surface membrane was labeled, as were membrane-bounded sporozoite organelles identified as micronemes. Observation by immunofluorescence, on sporozoites incubated with bovine peripheral blood lymphocytes, suggested that the antigen recognized by the monoclonal antibody does not enter the lymphocyte during sporozoite endocytosis. We conclude that synthesis of the antigen or its precursor(s) occurs during sporogony in the feeding tick, at the time of maximal parasite proliferation, and precedes the formation of morphologically mature sporozoites; the antigen's role in the parasite life cycle also appears to be limited to events associated with the sporozoite entry process. © 1985AcademicPress, Inc. INDEX DESCRIPTORS AND ABBREVIATIONS: Theileria parva; Protozoa, parasitic; Sporozoite, Antigen, surface; Rhipicephalus appendiculatus, Tick; Cryoultramicrotomy; Immunoautoradiography; Immunoelectron microscopy; Protein-A-colloidal gold; Immunofluorescence antibody test (IFA test); Monoclonal antibody specific for the T. parva sporozoite (MAbD1); Tritiated MAbD1 ([3H]MAbD1); Phosphate-buffered saline (PBS); Bovine serum albumin (BSA).
1 ~m in diameter, occurs during sporogony in the salivary gland cells of the tick and is stimulated by the tick feeding on a mammalian host (Martin et al. 1964; Purnell et al. 1973). Sporogony, resulting in up to 50,000 mature sporozoites per infected salivary gland acinus, is generally complete 35 days after the onset of tick feeding (Purnell and Joyner 1968; Fawcett et al. 1982a). When T. parva-infected ticks feed on susceptible cattle, mature sporozoites, inoculated with the tick saliva, infect bovine lymphocytes and develop into schizonts, which
INTRODUCTION
Sporozoites are the infective stage of the protozoan parasite, Theileria parva, which causes East Coast fever of cattle in East Africa. The disease is transmitted by the Ixodid tick, RhipicephaIus appendiculatus. Development and maturation of the parasite to infective sporozoites, approximately i Present address: Institut ft~r Genetik, KfK, Postfach 3640, D-7500 Karlsruhe, West Germany. z Present address: Veterinary Research Department of the Kenya Agricultural Research Institute, Muguga, Kenya. 90 0014-4894/85 $3.00 Copyright © 1985by AcademicPress, Inc. All rightsof reproductionin any form reserved.
TheiIeria parva: ANTIGEN EXPRESSION
are the pathogenic stage of the parasite. Mature sporozoites, harvested from ticks fed on rabbits for 4 days, can infect bovine lymphocytes in vitro (Brown et al. 1973)by an entry process described as passive endocytosis (Fawcett et al. 1982b).
Recently, the production of MAbD1, a monoclonal antibody that neutralized sporozoites from different T. p a r v a stocks, was reported (Dobbelaere et al. 1984b). The results of in vitro experiments on the sporozoite neutralizing activity of MAbD1 indicated that neutralization occurred by blocking sporozoite entry into the target cell rather than through antibody mediated lysis. MAbD1 detects a sporozoite surface antigen which has recently been identified by immunoprecipitation and immunoelectron microscopy: the antigen is a protein with a molecular weight of approximately 68,000 D and is distributed over the entire surface of the sporozoite (Dobbelaere et al. 1985). This sporozoite surface antigen is closely associated with the entry process of the sporozoite into the target cell (Webster et al. 1985). In the present study, other developmental stages of T. p a r v a were screened using an indirect IFA test for reactivity with MAbD1, and this led to the demonstration that the antigenic determinant is unique to the sporozoite stage. This study was also aimed at defining by immunoautoradiography and immunoelectron microscopy, the time and site of synthesis of the antigen or its precursors recognized by MAbDI in the sporoblast and to establish the fate of the sporozoite surface antigen after sporozoite entry into the target cell. MATERIALS AND METHODS MAbD1 was derived as described in detail by Dobbelaere et al. (1984b). Briefly, spleen cells from mice immunized with crude, freeze-thawed Theileria p a r v a sporozoite preparations were fused with X63-Ag8.653 myeloma cells (Kearney et al. 1979), and antibodyproducing cells were cloned by limiting dilution (Oi and Herzenberg 1980). MAbD1 is a Protein-A-binding IgG 3. Antibody was produced in ascites fluid as de-
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scribed by Hoogenraad et al. (1983) and used at different dilutions as specified below or purified first by adsorption on P r o t e i n - A - S e p h a r o s e CL-4B (Pharmacia, Uppsala, Sweden) by the method of Seppftl~i et al. (1981). For use in immunoautoradiography, biosynthetically tritiated MAbD1 was produced in ascites fluid according to the method of Dobbelaere and Spooner (in press). R h i p i c e p h a l u s a p p e n d i c u l a t u s nymphs were infected with T. p a r v a (Muguga) by feeding them on the ears of cattle infected with East Coast fever. The cattle had been infected by inoculation with cryopreserved s p o r o z o i t e s (Cunningham e t al. 1973) and were showing piroplasms in the blood at the time of tick application. Nymphs were allowed to moult to adults at 28 C, 85% relative humidity, and were stored for 13 months at 18 C, 85% relative humidity. To induce parasite maturation, adult ticks were fed on the ears of rabbits (New Zealand white) for either 2 or 4 days, depending on the experimental design. The location of the antigen or its precursor(s) in the developing T. p a r v a parasite or mature sporozoites was studied by cryoultramicrotomy. Protein-A-colloidal gold was used as an electron-dense label to detect binding of MAbD1. Salivary glands from 2- or 4day-fed ticks infected with T. p a r v a , that contained many infected acini, were selected and collected by the method described by Dobbelaere et al. (1984a). Glands were fixed in 4% formaldehyde in 100 mM phosphate buffer (pH 7.4) for 60 min at 22 C. After washing in the same buffer, they were embedded in 10% gelatin (Geuze and Slot 1980) and further processed as described by Webster et al. (1985). Tick hemolymph containing kinetes was obtained from the drops formed on the severed legs of 50 adult R . a p p e n d i c u l a t u s ticks (Young and Leitch 1980). The drops were smeared onto glass microscope slides, airdried, fixed in anhydrous acetone, and stored at - 2 0 C until use (Young and Leitch 1980). Bovine lymphoid cell lines, infected with schizonts from different T. p a r v a stocks, were cultured in vitro (Brown 1979) and used to prepare schizont antigen slides. The stocks tested were Muguga (Brocklesby et al. 1961), Kiambu 5 (Irvin et al. 1974), Uganda (Uilenberg 1981), Kilifi (Irvin et al. 1981b), Junju, Mavueni, Marikebuni, and Mariakani (Irvin et al. 1983). Antigen slides were prepared on Wellcome PTFE slides by a method similar to that described by Goddeeris et al. (1982) and adapted by Minani e t al. (1983) for screening of monoclonal antibodies raised against T. p a r v a schizonts. T. p a r v a merozoites were purified from a culture of bovine lymphoid cells infected with T. p a r v a (Kilifi), releasing free merozoites into the supernatant. Volumes of 100 ml of culture were spun at 80g for 5 min to remove the majority of cells, and the supernatant
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suspension was pelleted by centrifugation at 2500g for 20 min. The pellet was resuspended in 20 ml of PBS and filtered through an 8-p,m filter (Millipore, Bedford, MA, USA) to remove the remaining culture cells. The filtrate was concentrated by centrifugation at 2500g for 20 min and resuspended in 1.2 ml of Dulbecco's PBS (pH 7.4). Twelve cytospin preparations were made on microscope slides using a cytocentrifuge (Cytospin 2: Shandon Southern Instruments, Sewickley, England, UK). One hundred microliters of suspension per preparation was centrifuged at 660 rpm for 5 min. Eleven preparations were fixed in anhydrous acetone for 5 min and stored at - 2 0 C until use. One was fixed in methanol for 5 min, stained for 35 min in 5% Giemsa in distilled water, and examined by light microscopy at x 1000 magnification under oil immersion. The preparations contained 30 to 40 merozoites per field and no lymphoid cells. Piroplasm antigen preparations were prepared from the blood of a calf infected with T. parva at the height of piroplasm parasitosis (39% of the red blood cells infected). Antigen slides were prepared by the method of Burridge (1971). Sporozoites were mixed with bovine peripheral blood leucocytes, and samples were taken at different time intervals and examined by IFA test to determine whether the antigen recognized by MAbD1 could be found on sporozoites that had entered lymphocytes. A sporozoite preparation was made from 4-day-fed, T. parva-infected adult ticks (infection rate, 25 infected acini/tick). Salivary glands from 150 such ticks were removed aseptically and homogenized in a tissue grinder in 1 ml of Leibovitz's Lt5 medium (Gibco BioCult, Paisley, Scotland, UK) supplemented with 10% heat-inactivated fetal bovine serum, 10% tryptose phosphate broth, 20 fxg/ml L-glutamine, 100 IU/ml benzylpenicillin, 100~g/ml streptomycin sulfate, 50 p~g/ml gentamycin, and 5 x 10-SM 2-mercaptoethanol (complete L15). After centrifugation at 50g for 5 min, the supernatant was added to a pellet of 10 7 peripheral blood leucocytes collected from an East Coast feversusceptible cow and isolated on a Ficoll-sodium metrizoate gradient of specific gravity 1.077 (Fico11-Hypaque; Pharmacia) (Brown 1979). The leucocyte pellet was resuspended in the sporozoite suspension and incubated at 37 C for 1.5 hr. Thereafter, 1 ml of complete L15 was added, and the cells were transferred into one well of a 24-well tissue culture plate (Costar, Cambridge, MA, USA) for further incubation. Cytospin preparations of the leucocytes incubated with sporozoites were made on microscope slides using a cytocentrifuge (approximately 4 x 10 4 cells in 0.2 ml centrifuged at 660 rpm for 5 min). Cytospin preparations were made 10 min, 25 rain, 90 min, 4 hr, and 12 hr after peripheral blood leucocytes and sporozoites were mixed, and then every 6 hr for a total of 48 hr. Slides were then fixed in anhydrous acetone for 5 rain and stored at - 2 0 C.
T. parva schizont antigen prepared on slides was thawed and used immediately. Preparations of leucocytes and sporozoites, and kinete, merozoite, and piroplasm antigen preparations were thawed and delineated with a ring of nail varnish (1 cm diameter) before use. Schizont and piroplasm antigen preparations were incubated with 20 ~1 of MAbD1 ascites fluid, diluted 1:50 in PB S. Kinete antigen, merozoite antigen, and leucocyte-sporozoite preparations were incubated with Protein-A-purified MAbD1 (1 mg/ml) in PBS. Antibody was used at dilutions of 1:50 for kinete and merozoite antigen preparations and 1:250 for the leucocyte-sporozoite preparations. The preparations were incubated in moist chambers for 30 min at room temperature, after which they were washed three times in PBS. A further 30-min incubation was carried out after addition of 20 ~.1 of rabbit antimouse immunoglobulin conjugated with fluorescein isothiocyanate (Miles Laboratories, Slough, England, UK) at a dilution of 1:200 in PBS containing 0.01% Evans Blue. Slides were washed three times in PBS containing 1% BSA and mounted under a coverslip in 50% glycerol in PBS. They were then examined for fluorescence using an Orthoplan fluorescence microscope (Ernst Leitz, Wetzlar, West Germany) with x 6.3 eyepieces and x 63 or x 100 Phaco Fluorez objectives. Illumination was with a 200-W ultra-high-pressure mercury vapor lamp with 2 x KP490 and 1-mm GG455 excitation filters, a TK510 dichroic beam splitter, and a K515 suppression filter. Antigen preparations of tick salivary glands were obtained as described by Dobbelaere et al. (1984b) and consisted of 5-p.m cryostat sections of salivary glands from 75 T. parva-infected adult ticks fed on rabbits for 0, 1, 2, 3, or 4 days. Sections were fixed with 0.25% glutaraldehyde in distilled water for 5 min, washed once in PBS/BSA, and stored at - 8 0 C until use. Sections were thawed at room temperature, delineated with nail varnish, incubated for 45 min with 20 ~1 of [SH]MAbD1 ascites fluid diluted 1:250 in PBS, and washed three times with PBS/BSA. They were then stained with Feulgen DNAstain to reveal the parasite in sections of infected salivary gland acini. Slides were air-dried and dipped in Ilford L4 nuclear emulsion (I1ford, Basildon, Essex, England, UK), left to dry, exposed for 20 days at 4 C, and subsequently developed in Kodak D 76. After developing, slides were counterstained for 2 rain with Fast Green (1:500 dilution in distilled water of a stock solution containing 0.5% Fast Green, 0.3% phosphomolybdic acid, 0.3% phosphotungstic acid, 50% ethanol, and 5% acetic acid in distilled water). Sections were examined by light microscopy at x 500 magnification under oil immersion for the presence of silver grains over salivary gland acini containing sporozoite antigen, indicating binding of [3H]MAbD1. To compare the amount of sporozoite antigen present in sections of infected salivary gland acini from ticks fed for 0 to 4 days, silver grains per
Theileria parva: ANTIGEN EXPRESSION 154-1xm2 section were counted. These counts were done for 30 infected acini selected at random from two sections of each preparation. Background was calculated from the silver grain counts on 30 randomly selected uninfected acini from the same preparations. Results were statistically analyzed with S t u d e n t ' s t test. RESULTS
The IFA test with MAbD1 labeled Theileria parva sporozoites in leucocyte-sporozoite preparations but failed, at the dilutions tested, to label the kinete, schizont, merozoite, or piroplasm stages of the parasite. Immunoautoradiography on cryostat sections of tick salivary glands infected with T. parva revealed different degrees of labeling, depending on the maturity of the parasite (Fig. 1). Infected acini in ticks that had not fed were unlabeled or poorly labeled (Fig. 1A), but labeling became more pronounced on infected acini from 1- or 2day-fed ticks (Fig. 1B). Strong labeling of the parasite could be observed in infected acini from ticks that had fed for 3 or 4 days (Fig. 1C). The results of the silver grain counts are represented in Fig. 2. Although sections through infected salivary glands from ticks fed for 1 and 2 days already showed labeling with [3H]MAbDI, the most
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significant increase in silver grains on infected acini w a s observed in preparations from infected ticks that had fed for 3 days compared to preparations from ticks that fed for 2 days (P < 0.001). The increase in silver grains was accompanied by an increase in size of the infected acinus (Figs. 1 A - C ) , which also contained more Feulgen-positive material, reflecting an increase in D N A content of the infected acinus. The silver grain counts of different infected acini in any one preparation varied considerably, as reflected by the high standard deviations in Fig. 2. The lower counts were usually observed over infected acini which appeared to contain a less mature parasite, as judged by their Feulgen staining characteristics. The Fast Green counterstain made preparations easier to interpret, as it stained the cell cytoplasm green, thus delineating cells and making detection of sections of parasitized cells easier. This was especially the case for the weakly labeled, infected salivary gland acini from ticks fed for 0 or 1 day. Immunoelectron microscopic observations on ultrathin frozen sections of salivary glands from 2-day-fed infected ticks revealed labeling with Protein-A-colloidal gold of the sporoblast stage of the parasite
FIG. 1. Immunoautoradiographs of developing Theileriaparva during sporogony in salivary glands of feeding adult Rhipicephalus appendiculatus ticks. (Scale bar = 20 ~m). (1A) An unlabeled infected acinus from an unfed tick. (1B) A labeled infected acinus from a 2-day-fed tick. (1C) A strongly labeled infected acinus from a 4-day-fed tick.
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40
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36 32
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28 24 20
"~0~
12 8
4
O
1
2
3
4
Uninfected acinus
Days feeding
FIG. 2. Increase in antigenic determinants, recognized by the monoclonal antibody MAbD1, in The# leria parva-infected salivary gland acini of Rhipicephalus appendiculatus ticks after 0, 1, 2, 3, or 4 days feeding, measured by immunoautoradiography.
in infected E cells (Fig. 3). Sporozoite antigen, or its precursor(s), were detected in the infected cell, in the region occupied by parasite nuclei. There was scattered cytoplasmic labeling (Fig. 3A), and labeling of parasite membranes was observed, often including parasite nuclear membranes (Figs. 3B and C). Labeling of the labyrinth, an intricate network of anastomosing parasite cytoplasm processes intensely interwoven with the host cell cytoplasm, was not observed in these sections (not shown). Ultrathin frozen sections of salivary glands from 4-day-fed infected ticks incubated with MAbD1 showed labeling of the surface of mature sporozoites (Fig. 4A). MAbD1 was also found to label some membrane-bounded sporozoite organelles, identified as micronemes. In any one sporozoite, not all micronemes were labeled. Intraluminal (Fig. 4B) as well as microneme membrane labeling (Fig. 4C) was observed. The immunofluorescence observations on preparations of lymphocytes incubated with sporozoites can be summarized as follows: free fluorescent sporozoites could be seen, as well as fluorescing sporozoites at-
tached or closely associated with peripheral blood leucocytes (Fig. 5A); dome-shaped fluorescent structures, indicative of sporozoites in the process of entering lymphocytes, were also observed (Fig. 5B); fluorescent sporozoites apparently inside leucocytes were not seen. Occasionally, diffuse faint fluorescence could be seen on lymphocytes (Fig. 5C). Fluorescent patterns indicating sporozoite entry were predominantly present in preparations made during the first 90 min of incubation. Fluorescence in later preparations appeared to be restricted to free sporozoites or sporozoites that had attached to, but had not entered, lymphocytes. The faint scattered fluorescence on some lymphocytes was also present in these later preparations. Even after 48 hr, free fluorescent sporozoites could still be observed. DISCUSSION
The results of the IFA tests on different developmental stages of Theileria parva demonstrated that MAbD1 recognized a determinant on a sporozoite antigen which was not detectable, by the methods used, in the other stages of the parasite. These findings suggest that the antigen recognized by MAbD1 is a stage-specific antigen. A protective surface antigen of Plasmodium berghei sporozoites has also been shown to be stage specific (Aikawa et al. 1981). The fact that the kinete, which is the immediate precursor of the salivary gland stage of the parasite in the tick, was not detected by MAbD1 indicates that the sporozoite surface antigen and/or its precursor(s) carrying the corresponding determinant had not yet been synthesized. This was consistent with the observation that the next stage of the parasite, the sporoblast, which is found in the salivary glands of unfed adult ticks, was unlabeled or only poorly labeled in immunoautoradiographs (Fig. 1). In the present study, MAbD1 did not detect schizonts from bovine lymphoid cell
FIG. 3. Electron micrographs of ultrathin frozen sections of Theileria parva-infected salivary gland acini from 2-day-fed Rhipicephalus appendiculatus ticks, incubated with the monoclonal antibody MAbD1 followed by Protein-A-colloidal gold. (3A) Scattered colloidal gold labelling (arrows) of parasite cytoplasm in an infected E cell; a secretory granule (E) typical for the E cell is also shown. (Scale bar = 0.2 ~m). (3B) Demonstrates the presence of antigen or precursors of the antigen (arrows) associated with membranes of the parasite nucleus (pN). (Scale bar = 0.3 p,m). (3C) A high-magnification electron micrograph, showing labeling of parasite membranes (arrows). (Scale bar = 0.1 p~m). 95
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FIG. 4. Electron micrographs of ultrathin frozen sections of Theileria parva sporozoites in the salivary gland of Rhipicephalus appendiculatus ticks fed for 4 days, demonstrating labeling of the sporozoite surface coat as well as labeling of micronemes (M). (Scale bars = 0.1 ~m). (4A) Shows labeling of the peripheral membrane of the sporozoite, as well as labeling of some of the micronemes. (4B) Detail of intraluminal labeling of micronemes. (4C) Detail of labeling on the microneme membrane.
lines infected with T. parva by the IFA test. Furthermore, sporozoites could not be detected by immunofluorescence inside lymphocytes infected with sporozoites in vitro. Instead, the fluorescence patterns observed (Fig. 5) seemed to suggest that sporozoite surface antigen recognized by MAbD1 does not enter the cell during sporozoite endocytosis. A detailed immunoelectron microscopic study on the fate of the sporozoite surface antigen, detected by MAbD1, upon
sporozoite entry into bovine lymphocytes in vitro will be presented elsewhere (Webster et al. 1985). In the latter study, sporo-
zoites inside lymphocytes were unlabeled, but antigen was detected on the surface of target cells after entry or where contact between cells and sporozoites had occurred. The presence of sporozoite antigen on the surface of the lymphocyte may account for the faint diffuse fluorescence observed here.
Theileria parva: ANTIGEN EXPRESSION
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FIG. 5. Immunofluorescence patterns observed in acetone-fixed cytospin preparations of Theileria parva sporozoites incubated with bovine peripheral blood lymphocytes. (Scale bar = 5 p~m). (5A) A fluorescent sporozoite attached to a lymphocyte. (5B) Dome-shaped fluorescent pattern, indicative of a sporozoite in the process of entering a lymphocyte. (5C) Lymphocyte with sporozoite entering, also showing faint scattered fluorescence.
Schizonts are the stage of the T. parva parasite that develops in white blood cells after sporozoite infection. Fawcett et al. (1982b) and also Stagg et al. (1981) described c o n s i d e r a b l e m o r p h o l o g i c a l changes within the first 24 hr after entry of sporozoites into peripheral blood leucocytes in vitro. Monoclonal antibodies raised against schizonts can detect schizont antigen within 2 days after in vitro infection of bovine lymphoblasts with sporozoites (S. Black, personal communication). The apparent shedding of the sporozoite surface coat, in conjunction with the rapid disappearance of the typical sporozoite morphology, suggests that the sporozoite stage of the parasite and the antigens associated with it are shortlived in the infected host. No common determinants between the sporozoite and merozoite stages of T. parva were observed with MAbD1. Merozoites develop from the schizont stage in the lymphoblast, are released into circulation when the latter breaks up, and subsequently invade erythrocytes. The presence of common determinants on sporozoites and merozoites would not have been entirely surprising considering their morphological similarities and also considering the fact
that merozoites must also be equipped with a mechanism for recognition, attachment, and entry into ceils of bovine origin. It has been shown, in malaria for example, that a determinant recognized by monoclonal antibodies raised against cultured erythrocytic stages of P. falciparum is shared by both the sporozoite and the merozoite stages of the parasite (Hope et al. 1984). Immunoautoradiography showed that, although labeling of the parasite in 0-, 1-, or 2-day-fed ticks could be observed, most of the synthesis of the antigen recognized by MAbD1 occurred between the second and third days of tick feeding. This was reflected by the sudden increase in silver grain counts on infected acini of 3- or 4-dayfed ticks compared to counts for infected acini from 1- or 2-day-fed ticks (Fig. 2). The time of sporozoite surface antigen synthesis coincided with the incorporation of tritiated nucleic acid precursor(s) into parasite DNA and R N A during the development of the parasite in the feeding adult tick (Dobbelaere et al. 1983). This suggests that the sporozoite surface antigen or its precursor(s) are synthesized at the time when maximal parasite proliferation occurs. Immunoelectron microscopy on ultrathin
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frozen sections of infected salivary glands from 2-day-fed ticks, incubated with MAbD1, revealed labeling of sporoblasts with Protein-A-colloidal gold (Fig. 3). The intracellular label, observed in the region of parasite nuclear proliferation, may indicate sites of synthesis of the antigen or its precursor(s). This hypothesis is supported by the fact that parasite cytoplasm, associated with this region, was shown to include numerous mitochondria and to be rich in polyribosomes (Fawcett e t al. 1982a), organelles typically involved in protein synthesis. It was interesting to note that the labyrinth was unlabeled. This structure, intricately interwoven with host cell cytoplasm, may primarily be involved in absorption of precursor molecules through its plasmalemma (Fawcett et al. 1982a). The presence of Protein-A-colloidal gold on the surface of sporozoites (Fig. 4) from sections of salivary glands of 4-day-fed infected ticks confirmed the previous conclusion that MAbD1 recognizes a sporozoite surface antigen (Dobbelaere et al. 1985). The surface-labeling technique used in that study did not allow access to intracellular determinants recognized by MAbD1. The technique of ultrathin frozen sectioning used in the present work ensured total accessibility of MAbD1 to sites inside the parasite, and hence labeling of micronemes could be observed (Fig. 4). The presence of antigen associated with micronemes may indicate the involvement of these membrane-bounded organelles in transport of synthesized antigen to the parasite surface. Fawcett et al. (in press) reported that micronemes, during their formation, were first detected in the cytoplasm of the parasite, in close association with the nuclear envelope. This observation and the demonstration of antigen in the same region of the parasite, as well as on nuclear membranes, further supports our hypothesis that micronemes may be involved in transport of synthesized antigen to the parasite surface. Detection of P . k n o w l e s i sporozoite surface
antigen associated with micronemes has also been reported recently (Fine e t al. 1984). Light microscopic (Martin et al. 1964; Purnell and Joyner 1968; Purnell e t al. 1973; Irvin et al. 1981a) and electron microscopic (Fawcett et al. 1982a) data on sporogony of T. p a r v a in feeding adult ticks indicated that mature infective sporozoites are mainly produced 3 to 4 days after the tick starts feeding. The results of immunoautoradiography, the presence of intracellular labeling of sporoblasts, and the absence of internal label in sections through mature sporozoites strongly suggest that synthesis of the antigen recognized by MAbD1 precedes formation of morphologically mature sporozoites. The absence of an endoplasmic reticulum in sporozoites (Fawcett et aI. 1982a) also implies that membraneassociated components of the sporozoite surface would have to be synthesized and transported at an earlier stage in sporogony. This may also indicate that the small number of labeled micronemes observed in sporozoites may be remnants of a transport mechanism for parasite surface components, whereas the unlabeled micronemes observed in mature sporozoites may be responsible for secretion of other products, essential to the parasite at a later stage in the life cycle, e.g., dispersal of the host cell membrane after entry into the target cell (Fawcett et al. 1982b). The results of immunoautoradiography also indicated the time during development of the parasite at which the antigen is predominantly synthesized, and therefore the time at which the messenger RNA, involved in the synthesis of this antigen, can be expected to be present in infected salivary gland cells. Consequently, isolation of this messenger RNA would be optimal from ticks that have fed on rabbits for 2 to 3 days. In initial experiments at this laboratory, messenger RNA translation products specific for T. p a r v a have been synthesized in vitro from messenger RNA isolated from
Theileria parva: ANTIGEN EXPRESSION
salivary glands of T. parva-infected ticks fed on rabbits for 2.5 days (R.O. Williams, personal communication). MAbD1 recognizes a protein on the surface of the sporozoite and blocks entry of the sporozoite into the target cell (Dobbelaere et al. 1984b). MAbD1 must therefore bind to a molecule which may well have a unique function in the entry process, or is at least closely associated with sporozoite entry into the cell. The observations on synthesis of this antigen and the fact that the schizont developing after sporozoite entry is not detected by MAbD1 suggest that the antigen's involvement in the Theileria parva life cycle is limited to events associated with the sporozoite entry process. ACKNOWLEDGMENTS The authors thank J. N. Kiarie for excellent technical assistance. We also thank J. Katende for providing piroplasm antigen preparations and Dr. T. Minami for carrying out the IFA tests on Theileria parva schizont antigen preparations. Protein-A colloidal gold was a generous gift from Dr. G. Griffiths (European Molecular Biology Laboratory, Heidelberg, West Germany). D.A.E. Dobbelaere is supported by the Department of Development Cooperation of the Belgian Government. REFERENCES AIKAWA, M., YOSH1DA, N., NUSSENZWEIG, R. S., AND NUSSENZWEIG, V. 1981. The protective antigen of malarial sporozoites (Plasmodium berghei) is a differentiation antigen. Journal oflmmunology 126, 2494-2495. BROCKLESBY, D. W., BARNETT, S. F., AND SCOTT, G. R. 1961. Morbidity and mortality rates in East Coast fever (Theileria parva) infection and their application to drug screening procedures. British Veterinary Journal 117, 529-531. BROWN, C. G. D. 1979. Propagation of Theileria. In "Practical Tissue Culture Applications" (K. Maramorosch, and H. Hirumi, eds.), pp. 223-254. Academic Press, New York. BROWN, C. G. D., STAGG, D. A., PURNELL, R. E., KANHAI, G. K., AND PAYNE, R. C. 1973. Infection and transformation of bovine lymphoid cells in vitro by infective particles of Theileria parva. Nature (London) 245, 101-103. BURRIDGE, M. J. 1971. Application of the indirect flu-
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orescent antibody test in experimental East Coast fever (Theileria parva infection of cattle). Research in Veterinary Science 12, 338-341. CUNNINGHAM, M. E, BROWN, C. G. D., BURRIDGE, M. J., AND PURNELL, R. E. 1973. Cryopreservation of infective particles of Theileria parva. International Journal for Parasitology 3, 583- 587. DOBBELAERE, D. A. E., IRVIN, A. D., SPOONER, P. R., AND OCAMA, J. G. R. 1983. Incorporation of 3H hypoxanthine by Theileria parva during development in the feeding adult tick Rhipicephalus appendiculatus. International Journal for Parasitology 13, 107-112. DOBBELAERE, D. A. E., KIARIE, J. N., AND IRVIN, A. D. 1984a. A rapid method to select Rhipicephalus appendiculatus salivary glands infected with Theileria parva. Journal of Parasitology 70, 828829. DOBBELAERE, D. A. E., SHAPIRO, S. Z., AND WEBSTER, P. 1985. Identification of a surface antigen on Theileria parva sporozoites by monoclonal antibody: Proceedings of the National Academy of Science USA 82, 1771-1775. DOBBELAERE, D. A. E., AND SPOONER, P. R. Production in ascites fluid of biosynthetically labelled monoclonal antibody to Theileria parva sporozoites. Journal of lmmunological Methods, in press. DOBBELAERE, D. A. E., SPOONER, P. R., BARRY, W. C., AND IRVlN, A. D. 1984b. Monoclonal antibody neutralizes the sporozoite stage of different Theileria parva stocks. Parasite Immunology 6, 361-370. FAWCETT, D. W., BUSCHER, G., AND DOXSEY, S. 1982a. Salivary gland of the tick vector of East Coast fever. III The ultrastructure of sporogony in Theileria parva. Tissue and Cell 14, 183-206. FAWCETT, D. W., DOXSEY, S., STAGG, D. A., AND YOUNG, A. S. 1982b. The entry of sporozoites of Theileria parva into bovine lymphocytes in vitro: E l e c t r o n m i c r o s c o p i c o b s e r v a t i o n s . European Journal of Cell Biology 27, 10-21. FAWCETT, D. W., YOUNG, A. S., AND LEITCH, B. L. Sporogony in Theileria (Apicomplexa: Piroplasmida): A comparative ultrastructural study. Protozoology, in press. FINE, E., AIKAWA, M., COCHRANE, A. H., AND NUSSENZWEIG, R. S. 1984. I m m u n o - e l e c t r o n microscopic observations on Plasmodium knowlesi sporozoites: Localization of protective antigen and its precursors. American Journal of Tropical Medicine and Hygiene 33, 220-226. GEUZE, H. J., AND SLOT, J. W. 1980. Disproportional immunostaining patterns of two secretory proteins in guinea pig and rat exocrine pancreatic cells. An immunoferritin and fluorescence study. European Journal of Cell Biology, 21, 93-100. GODDEERIS, B. M., KATENDE, J. M., IRVIN, A. D., AND CHUMO, R. S. C. 1982. Indirect fluorescent an-
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tibody test for experimental epizootiological studies on East Coast fever (Theileria parva infection in cattle). Evaluation of cell culture schizont antigen fixed and stored in suspension. Research in Veterinary Science 33, 360-365. HOOGENRAAD, N., HELMAN, T., AND HOOGENRAAD, J. 1983. The effect of pre-injection of mice with pristane on ascites tumour formation and monoclonal antibody production. Journal o f I m m u n o l o g i c a l Methods 61, 317-320. HOPE, I. A., HALL, E R., SIMMONS, D. LL., HYDE, J. E., AND SCAIFE, J. G. 1984. Evidence for immunological cross-reaction between sporozoites and blood stages of a human malaria parasite. Nature (London) 308, 191-194. IRVIN, A. D., BOARER, C. D. H., DOBBELAERE, D. A. E., MAHAN, S. M., MASAKE, R., AND OCAMA, J. G. R. 1981a. Monitoring Theileria parva infection in adult Rhipicephalus appendiculatus. Parasitology 82, 137-147. IRVIN, A . D . , CHUMO, R. S. C., DOBBELAERE, D. A. E., GODDEER1S, B. M., KATENDE, J. M., MINAMI, T., OCAMA, J. G. R., AND'SPOONER, P. R. 1981b. Preliminary studies on East Coast fever in the Coast Province of Kenya. In "Advances in the Control of Theileriosis" (A. D. Irvin, M. P. Cunningham, and A. S. Young, eds.), pp. 66-70. Martinus Nijhof, The Hague. IRVIN, A. D., DOBBELAERE, D. A. E., MWAMACHI, D. M., MINAMI, T., SPOONER, P. R., AND OCAMA, J. G. R. 1983. I m m u n i z a t i o n against East Coast fever: Correlation between monoclonal antibody profiles of Theileria parva stocks and cross immunity in vivo. R e s e a r c h in Veterinary Science 35, 341-346. IRVlN, A. D., PURNELL, R. E., BROWN, C. G. D., CUNNINGHAM, M. P., LEDGER, M. A., AND PAYNE, R. C. 1974. The application of an indirect method of infecting ticks with piroplasms for the isolation of field infections. British Veterinary Journal 130, 280-287. KEARNEY, J. E, RADBRUCH, A., LIESEGANG, B., AND RAJEWSKY, K. 1979. A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines. Journal o f b n m u n o l o g y 123, 1548- t550.
MARTIN, H. M., BARNETT, S. E, AND VIDLER, B. O. 1964. Cyclic development and longevity of Theileria parva in the tick Rhipicephalus appendiculatus. Experimental Parasitology 15, 527-555. M1NANI, T., SPOONER, P. R., IRV1N, A. D., OCAMA, J. G. R., DOBBELAERE, D. A. E., AND FUJINAGA, T. 1983. Characterisation of stocks of Theileria parva by monoclonal antibody profiles. Research in Veterinary Science 35, 334-340. OI, V. T., AND HERZENBERG, L. A. 1980. Immunoglobulin-producing hybrid cell lines. In "Selected Methods in Cellular Immunology" (B. B. Mischell, and S. M. Shiigi, eds.), pp. 351-372. Freeman, San Francisco. PURNELL, R. E., BROWN, C. G. D., CUNNINGHAM, M . P . , BURRIDGE, M . J . , KIRIMI, I . M . , AND LEDGER, M. A. 1973. East Coast fever: Correlation between the morphology and infectivity of Theileria parva developing in its tick vector. Parasitology 66, 539-544. PURNELL, R. E., AND JOYNER, L. P. 1968. The development of Theileria parva in the salivary glands of the tick, Rhipicephalus appendieulatus. Parasitology 58, 725-732. SEPPAL~., I., SARVAS, H., Pt~TERFY, F., AND MAKEL.~, O. 1981. The four subclasses of IgG can be isolated from mouse serum by using Protein A-Sepharose. Scandinavian Journal o f Immunology 14, 335-342. STAGG, D. A., DOLAN, T. T., LEITCH, B. L., AND YOUNG, A. S. 1981. The initial stages of infection of cattle cells with Theileria parva sporozoites in vitro. Parasitology 83, 191-197. UILENBERG, G. 1981. Theilerial species of domestic livestock. In "Advances in the Control of Theileriosis" (A. D. Irvin, M. E Cunningham, and A. S. Young, eds.), pp. 4 - 3 7 . M a r t i n u s Nijhof, The Hague. WEBSTER, P., DOBBELAERE, O. A. E., AND FAWCETT, D. W. 1985. The entry of sporozoites of Theileria parva into bovine lymphocyte in vitro: Immunoelectronmicroscopic observations. European Journal o f Cell Biology 36, 157-162. YOUNG, A. S., AND LEITCH, B. L. 1980. A possible relationship between the development of Theileria species and the ecdysis of their tick hosts. Journal o f Parasitology 66, 356-359.
Theileria parva: Expression of a Sporozoite Surface Coat Antigen DIRK A. E. DOBBELAERE, 1 PAUL WEBSTER, BRIAN L. LEITCH, 2 W O L F P. VOIGT, AND A N T H O N Y D . IRVIN International Laboratory for Research on Animal Diseases, P.O. Box 30709, Nairobi, Kenya (Accepted for publication 11 December 1984) DOBBELAERE, D. A. E., WEBSTER, P., LEITCH, B. L., VOIGT, W. P., AND IRVIN, A. D. 1985. Theileria parva: Expression of a sporozoite surface coat antigen. Experimental Parasitology 60: 90-100. A monoclonal antibody specific for the Theileria parva sporozoite, which recognizes a determinant on the surface coat and blocks sporozoite infectivity, was used to investigate the presence of the determinant on other stages of the parasite lifecycle. Immunofluorescence techniques did not demonstrate this determinant on the kinete, schizont, merozoite, or piroplasm stages of the parasite. Immunoautoradiography, using a tritiated form of the monoclonal antibody, on sections of infected salivary glands collected from ticks that had fed for 0, 1, 2, 3, or 4 days revealed that the determinant recognized was synthesized predominantly during sporogony, between 2 to 3 days after the tick started feeding. Immunoelectron microscopy was performed on ultrathin frozen sections of infected tick salivary glands incubated with the monoclonal antibody followed by Protein-A-colloidal gold. The antigen or its precursor could be detected in the developing parasite. In ticks fed 2 days, the sporoblast was labeled, both in the cytoplasm and on parasite membranes, often including the nuclear envelope. In sections from ticks fed 4 days, the sporozoite surface membrane was labeled, as were membrane-bounded sporozoite organelles identified as micronemes. Observation by immunofluorescence, on sporozoites incubated with bovine peripheral blood lymphocytes, suggested that the antigen recognized by the monoclonal antibody does not enter the lymphocyte during sporozoite endocytosis. We conclude that synthesis of the antigen or its precursor(s) occurs during sporogony in the feeding tick, at the time of maximal parasite proliferation, and precedes the formation of morphologically mature sporozoites; the antigen's role in the parasite life cycle also appears to be limited to events associated with the sporozoite entry process. © 1985AcademicPress, Inc. INDEX DESCRIPTORS AND ABBREVIATIONS: Theileria parva; Protozoa, parasitic; Sporozoite, Antigen, surface; Rhipicephalus appendiculatus, Tick; Cryoultramicrotomy; Immunoautoradiography; Immunoelectron microscopy; Protein-A-colloidal gold; Immunofluorescence antibody test (IFA test); Monoclonal antibody specific for the T. parva sporozoite (MAbD1); Tritiated MAbD1 ([3H]MAbD1); Phosphate-buffered saline (PBS); Bovine serum albumin (BSA).
1 ~m in diameter, occurs during sporogony in the salivary gland cells of the tick and is stimulated by the tick feeding on a mammalian host (Martin et al. 1964; Purnell et al. 1973). Sporogony, resulting in up to 50,000 mature sporozoites per infected salivary gland acinus, is generally complete 35 days after the onset of tick feeding (Purnell and Joyner 1968; Fawcett et al. 1982a). When T. parva-infected ticks feed on susceptible cattle, mature sporozoites, inoculated with the tick saliva, infect bovine lymphocytes and develop into schizonts, which
INTRODUCTION
Sporozoites are the infective stage of the protozoan parasite, Theileria parva, which causes East Coast fever of cattle in East Africa. The disease is transmitted by the Ixodid tick, RhipicephaIus appendiculatus. Development and maturation of the parasite to infective sporozoites, approximately i Present address: Institut ft~r Genetik, KfK, Postfach 3640, D-7500 Karlsruhe, West Germany. z Present address: Veterinary Research Department of the Kenya Agricultural Research Institute, Muguga, Kenya. 90 0014-4894/85 $3.00 Copyright © 1985by AcademicPress, Inc. All rightsof reproductionin any form reserved.
TheiIeria parva: ANTIGEN EXPRESSION
are the pathogenic stage of the parasite. Mature sporozoites, harvested from ticks fed on rabbits for 4 days, can infect bovine lymphocytes in vitro (Brown et al. 1973)by an entry process described as passive endocytosis (Fawcett et al. 1982b).
Recently, the production of MAbD1, a monoclonal antibody that neutralized sporozoites from different T. p a r v a stocks, was reported (Dobbelaere et al. 1984b). The results of in vitro experiments on the sporozoite neutralizing activity of MAbD1 indicated that neutralization occurred by blocking sporozoite entry into the target cell rather than through antibody mediated lysis. MAbD1 detects a sporozoite surface antigen which has recently been identified by immunoprecipitation and immunoelectron microscopy: the antigen is a protein with a molecular weight of approximately 68,000 D and is distributed over the entire surface of the sporozoite (Dobbelaere et al. 1985). This sporozoite surface antigen is closely associated with the entry process of the sporozoite into the target cell (Webster et al. 1985). In the present study, other developmental stages of T. p a r v a were screened using an indirect IFA test for reactivity with MAbD1, and this led to the demonstration that the antigenic determinant is unique to the sporozoite stage. This study was also aimed at defining by immunoautoradiography and immunoelectron microscopy, the time and site of synthesis of the antigen or its precursors recognized by MAbDI in the sporoblast and to establish the fate of the sporozoite surface antigen after sporozoite entry into the target cell. MATERIALS AND METHODS MAbD1 was derived as described in detail by Dobbelaere et al. (1984b). Briefly, spleen cells from mice immunized with crude, freeze-thawed Theileria p a r v a sporozoite preparations were fused with X63-Ag8.653 myeloma cells (Kearney et al. 1979), and antibodyproducing cells were cloned by limiting dilution (Oi and Herzenberg 1980). MAbD1 is a Protein-A-binding IgG 3. Antibody was produced in ascites fluid as de-
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scribed by Hoogenraad et al. (1983) and used at different dilutions as specified below or purified first by adsorption on P r o t e i n - A - S e p h a r o s e CL-4B (Pharmacia, Uppsala, Sweden) by the method of Seppftl~i et al. (1981). For use in immunoautoradiography, biosynthetically tritiated MAbD1 was produced in ascites fluid according to the method of Dobbelaere and Spooner (in press). R h i p i c e p h a l u s a p p e n d i c u l a t u s nymphs were infected with T. p a r v a (Muguga) by feeding them on the ears of cattle infected with East Coast fever. The cattle had been infected by inoculation with cryopreserved s p o r o z o i t e s (Cunningham e t al. 1973) and were showing piroplasms in the blood at the time of tick application. Nymphs were allowed to moult to adults at 28 C, 85% relative humidity, and were stored for 13 months at 18 C, 85% relative humidity. To induce parasite maturation, adult ticks were fed on the ears of rabbits (New Zealand white) for either 2 or 4 days, depending on the experimental design. The location of the antigen or its precursor(s) in the developing T. p a r v a parasite or mature sporozoites was studied by cryoultramicrotomy. Protein-A-colloidal gold was used as an electron-dense label to detect binding of MAbD1. Salivary glands from 2- or 4day-fed ticks infected with T. p a r v a , that contained many infected acini, were selected and collected by the method described by Dobbelaere et al. (1984a). Glands were fixed in 4% formaldehyde in 100 mM phosphate buffer (pH 7.4) for 60 min at 22 C. After washing in the same buffer, they were embedded in 10% gelatin (Geuze and Slot 1980) and further processed as described by Webster et al. (1985). Tick hemolymph containing kinetes was obtained from the drops formed on the severed legs of 50 adult R . a p p e n d i c u l a t u s ticks (Young and Leitch 1980). The drops were smeared onto glass microscope slides, airdried, fixed in anhydrous acetone, and stored at - 2 0 C until use (Young and Leitch 1980). Bovine lymphoid cell lines, infected with schizonts from different T. p a r v a stocks, were cultured in vitro (Brown 1979) and used to prepare schizont antigen slides. The stocks tested were Muguga (Brocklesby et al. 1961), Kiambu 5 (Irvin et al. 1974), Uganda (Uilenberg 1981), Kilifi (Irvin et al. 1981b), Junju, Mavueni, Marikebuni, and Mariakani (Irvin et al. 1983). Antigen slides were prepared on Wellcome PTFE slides by a method similar to that described by Goddeeris et al. (1982) and adapted by Minani e t al. (1983) for screening of monoclonal antibodies raised against T. p a r v a schizonts. T. p a r v a merozoites were purified from a culture of bovine lymphoid cells infected with T. p a r v a (Kilifi), releasing free merozoites into the supernatant. Volumes of 100 ml of culture were spun at 80g for 5 min to remove the majority of cells, and the supernatant
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suspension was pelleted by centrifugation at 2500g for 20 min. The pellet was resuspended in 20 ml of PBS and filtered through an 8-p,m filter (Millipore, Bedford, MA, USA) to remove the remaining culture cells. The filtrate was concentrated by centrifugation at 2500g for 20 min and resuspended in 1.2 ml of Dulbecco's PBS (pH 7.4). Twelve cytospin preparations were made on microscope slides using a cytocentrifuge (Cytospin 2: Shandon Southern Instruments, Sewickley, England, UK). One hundred microliters of suspension per preparation was centrifuged at 660 rpm for 5 min. Eleven preparations were fixed in anhydrous acetone for 5 min and stored at - 2 0 C until use. One was fixed in methanol for 5 min, stained for 35 min in 5% Giemsa in distilled water, and examined by light microscopy at x 1000 magnification under oil immersion. The preparations contained 30 to 40 merozoites per field and no lymphoid cells. Piroplasm antigen preparations were prepared from the blood of a calf infected with T. parva at the height of piroplasm parasitosis (39% of the red blood cells infected). Antigen slides were prepared by the method of Burridge (1971). Sporozoites were mixed with bovine peripheral blood leucocytes, and samples were taken at different time intervals and examined by IFA test to determine whether the antigen recognized by MAbD1 could be found on sporozoites that had entered lymphocytes. A sporozoite preparation was made from 4-day-fed, T. parva-infected adult ticks (infection rate, 25 infected acini/tick). Salivary glands from 150 such ticks were removed aseptically and homogenized in a tissue grinder in 1 ml of Leibovitz's Lt5 medium (Gibco BioCult, Paisley, Scotland, UK) supplemented with 10% heat-inactivated fetal bovine serum, 10% tryptose phosphate broth, 20 fxg/ml L-glutamine, 100 IU/ml benzylpenicillin, 100~g/ml streptomycin sulfate, 50 p~g/ml gentamycin, and 5 x 10-SM 2-mercaptoethanol (complete L15). After centrifugation at 50g for 5 min, the supernatant was added to a pellet of 10 7 peripheral blood leucocytes collected from an East Coast feversusceptible cow and isolated on a Ficoll-sodium metrizoate gradient of specific gravity 1.077 (Fico11-Hypaque; Pharmacia) (Brown 1979). The leucocyte pellet was resuspended in the sporozoite suspension and incubated at 37 C for 1.5 hr. Thereafter, 1 ml of complete L15 was added, and the cells were transferred into one well of a 24-well tissue culture plate (Costar, Cambridge, MA, USA) for further incubation. Cytospin preparations of the leucocytes incubated with sporozoites were made on microscope slides using a cytocentrifuge (approximately 4 x 10 4 cells in 0.2 ml centrifuged at 660 rpm for 5 min). Cytospin preparations were made 10 min, 25 rain, 90 min, 4 hr, and 12 hr after peripheral blood leucocytes and sporozoites were mixed, and then every 6 hr for a total of 48 hr. Slides were then fixed in anhydrous acetone for 5 rain and stored at - 2 0 C.
T. parva schizont antigen prepared on slides was thawed and used immediately. Preparations of leucocytes and sporozoites, and kinete, merozoite, and piroplasm antigen preparations were thawed and delineated with a ring of nail varnish (1 cm diameter) before use. Schizont and piroplasm antigen preparations were incubated with 20 ~1 of MAbD1 ascites fluid, diluted 1:50 in PB S. Kinete antigen, merozoite antigen, and leucocyte-sporozoite preparations were incubated with Protein-A-purified MAbD1 (1 mg/ml) in PBS. Antibody was used at dilutions of 1:50 for kinete and merozoite antigen preparations and 1:250 for the leucocyte-sporozoite preparations. The preparations were incubated in moist chambers for 30 min at room temperature, after which they were washed three times in PBS. A further 30-min incubation was carried out after addition of 20 ~.1 of rabbit antimouse immunoglobulin conjugated with fluorescein isothiocyanate (Miles Laboratories, Slough, England, UK) at a dilution of 1:200 in PBS containing 0.01% Evans Blue. Slides were washed three times in PBS containing 1% BSA and mounted under a coverslip in 50% glycerol in PBS. They were then examined for fluorescence using an Orthoplan fluorescence microscope (Ernst Leitz, Wetzlar, West Germany) with x 6.3 eyepieces and x 63 or x 100 Phaco Fluorez objectives. Illumination was with a 200-W ultra-high-pressure mercury vapor lamp with 2 x KP490 and 1-mm GG455 excitation filters, a TK510 dichroic beam splitter, and a K515 suppression filter. Antigen preparations of tick salivary glands were obtained as described by Dobbelaere et al. (1984b) and consisted of 5-p.m cryostat sections of salivary glands from 75 T. parva-infected adult ticks fed on rabbits for 0, 1, 2, 3, or 4 days. Sections were fixed with 0.25% glutaraldehyde in distilled water for 5 min, washed once in PBS/BSA, and stored at - 8 0 C until use. Sections were thawed at room temperature, delineated with nail varnish, incubated for 45 min with 20 ~1 of [SH]MAbD1 ascites fluid diluted 1:250 in PBS, and washed three times with PBS/BSA. They were then stained with Feulgen DNAstain to reveal the parasite in sections of infected salivary gland acini. Slides were air-dried and dipped in Ilford L4 nuclear emulsion (I1ford, Basildon, Essex, England, UK), left to dry, exposed for 20 days at 4 C, and subsequently developed in Kodak D 76. After developing, slides were counterstained for 2 rain with Fast Green (1:500 dilution in distilled water of a stock solution containing 0.5% Fast Green, 0.3% phosphomolybdic acid, 0.3% phosphotungstic acid, 50% ethanol, and 5% acetic acid in distilled water). Sections were examined by light microscopy at x 500 magnification under oil immersion for the presence of silver grains over salivary gland acini containing sporozoite antigen, indicating binding of [3H]MAbD1. To compare the amount of sporozoite antigen present in sections of infected salivary gland acini from ticks fed for 0 to 4 days, silver grains per
Theileria parva: ANTIGEN EXPRESSION 154-1xm2 section were counted. These counts were done for 30 infected acini selected at random from two sections of each preparation. Background was calculated from the silver grain counts on 30 randomly selected uninfected acini from the same preparations. Results were statistically analyzed with S t u d e n t ' s t test. RESULTS
The IFA test with MAbD1 labeled Theileria parva sporozoites in leucocyte-sporozoite preparations but failed, at the dilutions tested, to label the kinete, schizont, merozoite, or piroplasm stages of the parasite. Immunoautoradiography on cryostat sections of tick salivary glands infected with T. parva revealed different degrees of labeling, depending on the maturity of the parasite (Fig. 1). Infected acini in ticks that had not fed were unlabeled or poorly labeled (Fig. 1A), but labeling became more pronounced on infected acini from 1- or 2day-fed ticks (Fig. 1B). Strong labeling of the parasite could be observed in infected acini from ticks that had fed for 3 or 4 days (Fig. 1C). The results of the silver grain counts are represented in Fig. 2. Although sections through infected salivary glands from ticks fed for 1 and 2 days already showed labeling with [3H]MAbDI, the most
93
significant increase in silver grains on infected acini w a s observed in preparations from infected ticks that had fed for 3 days compared to preparations from ticks that fed for 2 days (P < 0.001). The increase in silver grains was accompanied by an increase in size of the infected acinus (Figs. 1 A - C ) , which also contained more Feulgen-positive material, reflecting an increase in D N A content of the infected acinus. The silver grain counts of different infected acini in any one preparation varied considerably, as reflected by the high standard deviations in Fig. 2. The lower counts were usually observed over infected acini which appeared to contain a less mature parasite, as judged by their Feulgen staining characteristics. The Fast Green counterstain made preparations easier to interpret, as it stained the cell cytoplasm green, thus delineating cells and making detection of sections of parasitized cells easier. This was especially the case for the weakly labeled, infected salivary gland acini from ticks fed for 0 or 1 day. Immunoelectron microscopic observations on ultrathin frozen sections of salivary glands from 2-day-fed infected ticks revealed labeling with Protein-A-colloidal gold of the sporoblast stage of the parasite
FIG. 1. Immunoautoradiographs of developing Theileriaparva during sporogony in salivary glands of feeding adult Rhipicephalus appendiculatus ticks. (Scale bar = 20 ~m). (1A) An unlabeled infected acinus from an unfed tick. (1B) A labeled infected acinus from a 2-day-fed tick. (1C) A strongly labeled infected acinus from a 4-day-fed tick.
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DOBBELAERE ET AL.
40
•~
36 32
,'_c z
28 24 20
"~0~
12 8
4
O
1
2
3
4
Uninfected acinus
Days feeding
FIG. 2. Increase in antigenic determinants, recognized by the monoclonal antibody MAbD1, in The# leria parva-infected salivary gland acini of Rhipicephalus appendiculatus ticks after 0, 1, 2, 3, or 4 days feeding, measured by immunoautoradiography.
in infected E cells (Fig. 3). Sporozoite antigen, or its precursor(s), were detected in the infected cell, in the region occupied by parasite nuclei. There was scattered cytoplasmic labeling (Fig. 3A), and labeling of parasite membranes was observed, often including parasite nuclear membranes (Figs. 3B and C). Labeling of the labyrinth, an intricate network of anastomosing parasite cytoplasm processes intensely interwoven with the host cell cytoplasm, was not observed in these sections (not shown). Ultrathin frozen sections of salivary glands from 4-day-fed infected ticks incubated with MAbD1 showed labeling of the surface of mature sporozoites (Fig. 4A). MAbD1 was also found to label some membrane-bounded sporozoite organelles, identified as micronemes. In any one sporozoite, not all micronemes were labeled. Intraluminal (Fig. 4B) as well as microneme membrane labeling (Fig. 4C) was observed. The immunofluorescence observations on preparations of lymphocytes incubated with sporozoites can be summarized as follows: free fluorescent sporozoites could be seen, as well as fluorescing sporozoites at-
tached or closely associated with peripheral blood leucocytes (Fig. 5A); dome-shaped fluorescent structures, indicative of sporozoites in the process of entering lymphocytes, were also observed (Fig. 5B); fluorescent sporozoites apparently inside leucocytes were not seen. Occasionally, diffuse faint fluorescence could be seen on lymphocytes (Fig. 5C). Fluorescent patterns indicating sporozoite entry were predominantly present in preparations made during the first 90 min of incubation. Fluorescence in later preparations appeared to be restricted to free sporozoites or sporozoites that had attached to, but had not entered, lymphocytes. The faint scattered fluorescence on some lymphocytes was also present in these later preparations. Even after 48 hr, free fluorescent sporozoites could still be observed. DISCUSSION
The results of the IFA tests on different developmental stages of Theileria parva demonstrated that MAbD1 recognized a determinant on a sporozoite antigen which was not detectable, by the methods used, in the other stages of the parasite. These findings suggest that the antigen recognized by MAbD1 is a stage-specific antigen. A protective surface antigen of Plasmodium berghei sporozoites has also been shown to be stage specific (Aikawa et al. 1981). The fact that the kinete, which is the immediate precursor of the salivary gland stage of the parasite in the tick, was not detected by MAbD1 indicates that the sporozoite surface antigen and/or its precursor(s) carrying the corresponding determinant had not yet been synthesized. This was consistent with the observation that the next stage of the parasite, the sporoblast, which is found in the salivary glands of unfed adult ticks, was unlabeled or only poorly labeled in immunoautoradiographs (Fig. 1). In the present study, MAbD1 did not detect schizonts from bovine lymphoid cell
FIG. 3. Electron micrographs of ultrathin frozen sections of Theileria parva-infected salivary gland acini from 2-day-fed Rhipicephalus appendiculatus ticks, incubated with the monoclonal antibody MAbD1 followed by Protein-A-colloidal gold. (3A) Scattered colloidal gold labelling (arrows) of parasite cytoplasm in an infected E cell; a secretory granule (E) typical for the E cell is also shown. (Scale bar = 0.2 ~m). (3B) Demonstrates the presence of antigen or precursors of the antigen (arrows) associated with membranes of the parasite nucleus (pN). (Scale bar = 0.3 p,m). (3C) A high-magnification electron micrograph, showing labeling of parasite membranes (arrows). (Scale bar = 0.1 p~m). 95
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FIG. 4. Electron micrographs of ultrathin frozen sections of Theileria parva sporozoites in the salivary gland of Rhipicephalus appendiculatus ticks fed for 4 days, demonstrating labeling of the sporozoite surface coat as well as labeling of micronemes (M). (Scale bars = 0.1 ~m). (4A) Shows labeling of the peripheral membrane of the sporozoite, as well as labeling of some of the micronemes. (4B) Detail of intraluminal labeling of micronemes. (4C) Detail of labeling on the microneme membrane.
lines infected with T. parva by the IFA test. Furthermore, sporozoites could not be detected by immunofluorescence inside lymphocytes infected with sporozoites in vitro. Instead, the fluorescence patterns observed (Fig. 5) seemed to suggest that sporozoite surface antigen recognized by MAbD1 does not enter the cell during sporozoite endocytosis. A detailed immunoelectron microscopic study on the fate of the sporozoite surface antigen, detected by MAbD1, upon
sporozoite entry into bovine lymphocytes in vitro will be presented elsewhere (Webster et al. 1985). In the latter study, sporo-
zoites inside lymphocytes were unlabeled, but antigen was detected on the surface of target cells after entry or where contact between cells and sporozoites had occurred. The presence of sporozoite antigen on the surface of the lymphocyte may account for the faint diffuse fluorescence observed here.
Theileria parva: ANTIGEN EXPRESSION
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FIG. 5. Immunofluorescence patterns observed in acetone-fixed cytospin preparations of Theileria parva sporozoites incubated with bovine peripheral blood lymphocytes. (Scale bar = 5 p~m). (5A) A fluorescent sporozoite attached to a lymphocyte. (5B) Dome-shaped fluorescent pattern, indicative of a sporozoite in the process of entering a lymphocyte. (5C) Lymphocyte with sporozoite entering, also showing faint scattered fluorescence.
Schizonts are the stage of the T. parva parasite that develops in white blood cells after sporozoite infection. Fawcett et al. (1982b) and also Stagg et al. (1981) described c o n s i d e r a b l e m o r p h o l o g i c a l changes within the first 24 hr after entry of sporozoites into peripheral blood leucocytes in vitro. Monoclonal antibodies raised against schizonts can detect schizont antigen within 2 days after in vitro infection of bovine lymphoblasts with sporozoites (S. Black, personal communication). The apparent shedding of the sporozoite surface coat, in conjunction with the rapid disappearance of the typical sporozoite morphology, suggests that the sporozoite stage of the parasite and the antigens associated with it are shortlived in the infected host. No common determinants between the sporozoite and merozoite stages of T. parva were observed with MAbD1. Merozoites develop from the schizont stage in the lymphoblast, are released into circulation when the latter breaks up, and subsequently invade erythrocytes. The presence of common determinants on sporozoites and merozoites would not have been entirely surprising considering their morphological similarities and also considering the fact
that merozoites must also be equipped with a mechanism for recognition, attachment, and entry into ceils of bovine origin. It has been shown, in malaria for example, that a determinant recognized by monoclonal antibodies raised against cultured erythrocytic stages of P. falciparum is shared by both the sporozoite and the merozoite stages of the parasite (Hope et al. 1984). Immunoautoradiography showed that, although labeling of the parasite in 0-, 1-, or 2-day-fed ticks could be observed, most of the synthesis of the antigen recognized by MAbD1 occurred between the second and third days of tick feeding. This was reflected by the sudden increase in silver grain counts on infected acini of 3- or 4-dayfed ticks compared to counts for infected acini from 1- or 2-day-fed ticks (Fig. 2). The time of sporozoite surface antigen synthesis coincided with the incorporation of tritiated nucleic acid precursor(s) into parasite DNA and R N A during the development of the parasite in the feeding adult tick (Dobbelaere et al. 1983). This suggests that the sporozoite surface antigen or its precursor(s) are synthesized at the time when maximal parasite proliferation occurs. Immunoelectron microscopy on ultrathin
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frozen sections of infected salivary glands from 2-day-fed ticks, incubated with MAbD1, revealed labeling of sporoblasts with Protein-A-colloidal gold (Fig. 3). The intracellular label, observed in the region of parasite nuclear proliferation, may indicate sites of synthesis of the antigen or its precursor(s). This hypothesis is supported by the fact that parasite cytoplasm, associated with this region, was shown to include numerous mitochondria and to be rich in polyribosomes (Fawcett e t al. 1982a), organelles typically involved in protein synthesis. It was interesting to note that the labyrinth was unlabeled. This structure, intricately interwoven with host cell cytoplasm, may primarily be involved in absorption of precursor molecules through its plasmalemma (Fawcett et al. 1982a). The presence of Protein-A-colloidal gold on the surface of sporozoites (Fig. 4) from sections of salivary glands of 4-day-fed infected ticks confirmed the previous conclusion that MAbD1 recognizes a sporozoite surface antigen (Dobbelaere et al. 1985). The surface-labeling technique used in that study did not allow access to intracellular determinants recognized by MAbD1. The technique of ultrathin frozen sectioning used in the present work ensured total accessibility of MAbD1 to sites inside the parasite, and hence labeling of micronemes could be observed (Fig. 4). The presence of antigen associated with micronemes may indicate the involvement of these membrane-bounded organelles in transport of synthesized antigen to the parasite surface. Fawcett et al. (in press) reported that micronemes, during their formation, were first detected in the cytoplasm of the parasite, in close association with the nuclear envelope. This observation and the demonstration of antigen in the same region of the parasite, as well as on nuclear membranes, further supports our hypothesis that micronemes may be involved in transport of synthesized antigen to the parasite surface. Detection of P . k n o w l e s i sporozoite surface
antigen associated with micronemes has also been reported recently (Fine e t al. 1984). Light microscopic (Martin et al. 1964; Purnell and Joyner 1968; Purnell e t al. 1973; Irvin et al. 1981a) and electron microscopic (Fawcett et al. 1982a) data on sporogony of T. p a r v a in feeding adult ticks indicated that mature infective sporozoites are mainly produced 3 to 4 days after the tick starts feeding. The results of immunoautoradiography, the presence of intracellular labeling of sporoblasts, and the absence of internal label in sections through mature sporozoites strongly suggest that synthesis of the antigen recognized by MAbD1 precedes formation of morphologically mature sporozoites. The absence of an endoplasmic reticulum in sporozoites (Fawcett et aI. 1982a) also implies that membraneassociated components of the sporozoite surface would have to be synthesized and transported at an earlier stage in sporogony. This may also indicate that the small number of labeled micronemes observed in sporozoites may be remnants of a transport mechanism for parasite surface components, whereas the unlabeled micronemes observed in mature sporozoites may be responsible for secretion of other products, essential to the parasite at a later stage in the life cycle, e.g., dispersal of the host cell membrane after entry into the target cell (Fawcett et al. 1982b). The results of immunoautoradiography also indicated the time during development of the parasite at which the antigen is predominantly synthesized, and therefore the time at which the messenger RNA, involved in the synthesis of this antigen, can be expected to be present in infected salivary gland cells. Consequently, isolation of this messenger RNA would be optimal from ticks that have fed on rabbits for 2 to 3 days. In initial experiments at this laboratory, messenger RNA translation products specific for T. p a r v a have been synthesized in vitro from messenger RNA isolated from
Theileria parva: ANTIGEN EXPRESSION
salivary glands of T. parva-infected ticks fed on rabbits for 2.5 days (R.O. Williams, personal communication). MAbD1 recognizes a protein on the surface of the sporozoite and blocks entry of the sporozoite into the target cell (Dobbelaere et al. 1984b). MAbD1 must therefore bind to a molecule which may well have a unique function in the entry process, or is at least closely associated with sporozoite entry into the cell. The observations on synthesis of this antigen and the fact that the schizont developing after sporozoite entry is not detected by MAbD1 suggest that the antigen's involvement in the Theileria parva life cycle is limited to events associated with the sporozoite entry process. ACKNOWLEDGMENTS The authors thank J. N. Kiarie for excellent technical assistance. We also thank J. Katende for providing piroplasm antigen preparations and Dr. T. Minami for carrying out the IFA tests on Theileria parva schizont antigen preparations. Protein-A colloidal gold was a generous gift from Dr. G. Griffiths (European Molecular Biology Laboratory, Heidelberg, West Germany). D.A.E. Dobbelaere is supported by the Department of Development Cooperation of the Belgian Government. REFERENCES AIKAWA, M., YOSH1DA, N., NUSSENZWEIG, R. S., AND NUSSENZWEIG, V. 1981. The protective antigen of malarial sporozoites (Plasmodium berghei) is a differentiation antigen. Journal oflmmunology 126, 2494-2495. BROCKLESBY, D. W., BARNETT, S. F., AND SCOTT, G. R. 1961. Morbidity and mortality rates in East Coast fever (Theileria parva) infection and their application to drug screening procedures. British Veterinary Journal 117, 529-531. BROWN, C. G. D. 1979. Propagation of Theileria. In "Practical Tissue Culture Applications" (K. Maramorosch, and H. Hirumi, eds.), pp. 223-254. Academic Press, New York. BROWN, C. G. D., STAGG, D. A., PURNELL, R. E., KANHAI, G. K., AND PAYNE, R. C. 1973. Infection and transformation of bovine lymphoid cells in vitro by infective particles of Theileria parva. Nature (London) 245, 101-103. BURRIDGE, M. J. 1971. Application of the indirect flu-
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orescent antibody test in experimental East Coast fever (Theileria parva infection of cattle). Research in Veterinary Science 12, 338-341. CUNNINGHAM, M. E, BROWN, C. G. D., BURRIDGE, M. J., AND PURNELL, R. E. 1973. Cryopreservation of infective particles of Theileria parva. International Journal for Parasitology 3, 583- 587. DOBBELAERE, D. A. E., IRVIN, A. D., SPOONER, P. R., AND OCAMA, J. G. R. 1983. Incorporation of 3H hypoxanthine by Theileria parva during development in the feeding adult tick Rhipicephalus appendiculatus. International Journal for Parasitology 13, 107-112. DOBBELAERE, D. A. E., KIARIE, J. N., AND IRVIN, A. D. 1984a. A rapid method to select Rhipicephalus appendiculatus salivary glands infected with Theileria parva. Journal of Parasitology 70, 828829. DOBBELAERE, D. A. E., SHAPIRO, S. Z., AND WEBSTER, P. 1985. Identification of a surface antigen on Theileria parva sporozoites by monoclonal antibody: Proceedings of the National Academy of Science USA 82, 1771-1775. DOBBELAERE, D. A. E., AND SPOONER, P. R. Production in ascites fluid of biosynthetically labelled monoclonal antibody to Theileria parva sporozoites. Journal of lmmunological Methods, in press. DOBBELAERE, D. A. E., SPOONER, P. R., BARRY, W. C., AND IRVlN, A. D. 1984b. Monoclonal antibody neutralizes the sporozoite stage of different Theileria parva stocks. Parasite Immunology 6, 361-370. FAWCETT, D. W., BUSCHER, G., AND DOXSEY, S. 1982a. Salivary gland of the tick vector of East Coast fever. III The ultrastructure of sporogony in Theileria parva. Tissue and Cell 14, 183-206. FAWCETT, D. W., DOXSEY, S., STAGG, D. A., AND YOUNG, A. S. 1982b. The entry of sporozoites of Theileria parva into bovine lymphocytes in vitro: E l e c t r o n m i c r o s c o p i c o b s e r v a t i o n s . European Journal of Cell Biology 27, 10-21. FAWCETT, D. W., YOUNG, A. S., AND LEITCH, B. L. Sporogony in Theileria (Apicomplexa: Piroplasmida): A comparative ultrastructural study. Protozoology, in press. FINE, E., AIKAWA, M., COCHRANE, A. H., AND NUSSENZWEIG, R. S. 1984. I m m u n o - e l e c t r o n microscopic observations on Plasmodium knowlesi sporozoites: Localization of protective antigen and its precursors. American Journal of Tropical Medicine and Hygiene 33, 220-226. GEUZE, H. J., AND SLOT, J. W. 1980. Disproportional immunostaining patterns of two secretory proteins in guinea pig and rat exocrine pancreatic cells. An immunoferritin and fluorescence study. European Journal of Cell Biology, 21, 93-100. GODDEERIS, B. M., KATENDE, J. M., IRVIN, A. D., AND CHUMO, R. S. C. 1982. Indirect fluorescent an-
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tibody test for experimental epizootiological studies on East Coast fever (Theileria parva infection in cattle). Evaluation of cell culture schizont antigen fixed and stored in suspension. Research in Veterinary Science 33, 360-365. HOOGENRAAD, N., HELMAN, T., AND HOOGENRAAD, J. 1983. The effect of pre-injection of mice with pristane on ascites tumour formation and monoclonal antibody production. Journal o f I m m u n o l o g i c a l Methods 61, 317-320. HOPE, I. A., HALL, E R., SIMMONS, D. LL., HYDE, J. E., AND SCAIFE, J. G. 1984. Evidence for immunological cross-reaction between sporozoites and blood stages of a human malaria parasite. Nature (London) 308, 191-194. IRVIN, A. D., BOARER, C. D. H., DOBBELAERE, D. A. E., MAHAN, S. M., MASAKE, R., AND OCAMA, J. G. R. 1981a. Monitoring Theileria parva infection in adult Rhipicephalus appendiculatus. Parasitology 82, 137-147. IRVIN, A . D . , CHUMO, R. S. C., DOBBELAERE, D. A. E., GODDEER1S, B. M., KATENDE, J. M., MINAMI, T., OCAMA, J. G. R., AND'SPOONER, P. R. 1981b. Preliminary studies on East Coast fever in the Coast Province of Kenya. In "Advances in the Control of Theileriosis" (A. D. Irvin, M. P. Cunningham, and A. S. Young, eds.), pp. 66-70. Martinus Nijhof, The Hague. IRVIN, A. D., DOBBELAERE, D. A. E., MWAMACHI, D. M., MINAMI, T., SPOONER, P. R., AND OCAMA, J. G. R. 1983. I m m u n i z a t i o n against East Coast fever: Correlation between monoclonal antibody profiles of Theileria parva stocks and cross immunity in vivo. R e s e a r c h in Veterinary Science 35, 341-346. IRVlN, A. D., PURNELL, R. E., BROWN, C. G. D., CUNNINGHAM, M. P., LEDGER, M. A., AND PAYNE, R. C. 1974. The application of an indirect method of infecting ticks with piroplasms for the isolation of field infections. British Veterinary Journal 130, 280-287. KEARNEY, J. E, RADBRUCH, A., LIESEGANG, B., AND RAJEWSKY, K. 1979. A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines. Journal o f b n m u n o l o g y 123, 1548- t550.
MARTIN, H. M., BARNETT, S. E, AND VIDLER, B. O. 1964. Cyclic development and longevity of Theileria parva in the tick Rhipicephalus appendiculatus. Experimental Parasitology 15, 527-555. M1NANI, T., SPOONER, P. R., IRV1N, A. D., OCAMA, J. G. R., DOBBELAERE, D. A. E., AND FUJINAGA, T. 1983. Characterisation of stocks of Theileria parva by monoclonal antibody profiles. Research in Veterinary Science 35, 334-340. OI, V. T., AND HERZENBERG, L. A. 1980. Immunoglobulin-producing hybrid cell lines. In "Selected Methods in Cellular Immunology" (B. B. Mischell, and S. M. Shiigi, eds.), pp. 351-372. Freeman, San Francisco. PURNELL, R. E., BROWN, C. G. D., CUNNINGHAM, M . P . , BURRIDGE, M . J . , KIRIMI, I . M . , AND LEDGER, M. A. 1973. East Coast fever: Correlation between the morphology and infectivity of Theileria parva developing in its tick vector. Parasitology 66, 539-544. PURNELL, R. E., AND JOYNER, L. P. 1968. The development of Theileria parva in the salivary glands of the tick, Rhipicephalus appendieulatus. Parasitology 58, 725-732. SEPPAL~., I., SARVAS, H., Pt~TERFY, F., AND MAKEL.~, O. 1981. The four subclasses of IgG can be isolated from mouse serum by using Protein A-Sepharose. Scandinavian Journal o f Immunology 14, 335-342. STAGG, D. A., DOLAN, T. T., LEITCH, B. L., AND YOUNG, A. S. 1981. The initial stages of infection of cattle cells with Theileria parva sporozoites in vitro. Parasitology 83, 191-197. UILENBERG, G. 1981. Theilerial species of domestic livestock. In "Advances in the Control of Theileriosis" (A. D. Irvin, M. E Cunningham, and A. S. Young, eds.), pp. 4 - 3 7 . M a r t i n u s Nijhof, The Hague. WEBSTER, P., DOBBELAERE, O. A. E., AND FAWCETT, D. W. 1985. The entry of sporozoites of Theileria parva into bovine lymphocyte in vitro: Immunoelectronmicroscopic observations. European Journal o f Cell Biology 36, 157-162. YOUNG, A. S., AND LEITCH, B. L. 1980. A possible relationship between the development of Theileria species and the ecdysis of their tick hosts. Journal o f Parasitology 66, 356-359.