Proc. Nati. Acad. Sci. USAVol. 86, pp. 4639-4643, June 1989Immunology

Theileria annulata sporozoite surface antigen expressed inEscherichia coli elicits neutralizing antibody

(monoclonal antibody/vaccine/antigen gene/fusion protein/protozoan parasite)

SUSANNA WILLIAMSON*, ANDREW TAITt, DUNCAN BROWN*, ALAN WALKER*, PETER BECKt,BRIAN SHIELSt, JUNE FLETCHER*, AND ROGER HALLt**Centre for Tropical Veterinary Medicine, University of Edinburgh, Easter Bush, Roslin, Midlothian, EH25 9RG, Scotland, United Kingdom; and tWellcomeUnit for Molecular Parasitology, University of Glasgow, Garscube Estate, Bearsden, Glasgow, G61 1QH, Scotland, United Kingdom

Communicated by Don W. Fawcett, March 6, 1989

ABSTRACT Theileria annulata is an economically impor-tant protozoan parasite that threatens an estimated 250 millioncattle with the disease tropical theileriosis. Development of adefmed subunit vaccine is one means of trying to developcontrol measures against the disease. To this end we havecharacterized a surface antigen complex of the infective stage(sporozoite), by using a monoclonal antibody that neutralizessporozoite infectivity in vitro. We have cloned the gene codingfor this complex and have demonstrated that a fusion proteinexpressed from a fragment of this gene elicits strong neutral-izing antibodies. Furthermore we provide data on the structureand expression of this gene. In particular we show that theregion of the gene, expressed in one clone, codes for a proteinsegment relatively rich in proline residues. Also we demon-strate that expression of this gene appears to be stage specific,transcripts being present only in the sporoblast and sporozoitestages. The relevance of these rmdings to the production of adefined subunit vaccine is discussed.

Tropical theileriosis threatens an estimated 250 million cattlein the Mediterranean littoral, North Africa, the Middle East,the Indian subcontinent, and Central Asia and acts as a majorconstraint on livestock production and improvement in manydeveloping countries (1). The disease is caused by a tick-borne protozoan, Theileria annulata, which enters the bovinehost as the infective sporozoite stage in the saliva of a feedingtick. These sporozoites rapidly invade mononuclear cells inwhich the macroschizont stage develops; subsequently theseparasites are liberated, whereupon they invade erythrocytesproducing the piroplasm stage of the life cycle (2). Presentmethods of vaccination depend upon establishing activeinfection (3) and thus incur both the risk of causing clinicaldisease and the problems of handling live parasite materialunder tropical conditions. For these reasons we are activelyengaged in identifying parasite antigens capable of inducinga protective immune response (4-6) using similar approachesto those followed by investigators working on Theileriaparva, the cause ofEast Coast fever (7-11). Here we describean antisporozoite monoclonal antibody (mAb) that exhibitssignificant sporozoite neutralizing activity and provide dataon the nature of the polypeptides recognized by this anti-body. In addition we describe the isolation of two recombi-nant DNA clones that express the epitope defined by thismAb and we report our findings on the structure and expres-sion of this gene(s).§

MATERIALS AND METHODSParasite Material. Three uncloned isolates of Theileria

annulata were used in this study: they are called Ankara,

from Turkey (12); Hissar, from India (13); and Gharb, fromMorocco (14). Sporozoites for mAb production, immuno-fluorescence tests, inhibition assays, and immunoblottingwere obtained from Theileria annulata-infected adult ticks(Hyalomma anatolicum anatolicum). These ticks were fed onrabbits for 3 days to stimulate sporozoite maturation (15) afterwhich they were ground up and the sporozoites were col-lected in the form of an 8-,um filtrate of the supernatant asdescribed (16). Sporozoite RNA was extracted from infectedsalivary glands dissected from adult ticks after a 2-day feedon rabbits. Infected adult ticks were produced by allowingnymphs to engorge with blood on an infected calf exhibitinga patent piroplasm parasitaemia. Blood was also taken at thisstage of an experimentally induced infection in order toobtain piroplasms. This blood was first depleted of leuko-cytes (17) and then subjected to ammonium chloride lysis (18)in order to release the piroplasms, which were then harvestedand subjected to further manipulations as required. Thesepurified piroplasms routinely had contaminating lympho-cytes present at <0.001%. Hissar macroschizonts were main-tained in a bovine lymphosarcoma cell line [called BL-20 (19)]in tissue culture in RPMI medium plus 15% heat-inactivatedfetal calf serum at 37TC in 5% CO2 in air as described (16);uninfected BL-20 was maintained under identical conditions.mAb Production. Theileria annulata (Ankara) sporozoites

were purified from ground-up tick supernatant filtrate bycentrifugation on discontinuous Percoll (Pharmacia) densitygradients as described (20). A BALB/c mouse was inoculatedintraperitoneally with 6, 16, and 16 tick equivalents of livepurified sporozoites on days 0, 14, and 42, respectively.Three days after a final intravenous inoculation of 4 tickequivalents of sporozoites on day 204, the mouse was killed,its spleen was removed, and splenocytes were fused withX63-Ag8.653 myeloma cells (21) by a method based on astandard protocol (22). Hybridoma cultures secreting anti-sporozoite antibody, as detected by indirect immunofluores-cence on formalin-fixed sporozoites (4), were selected forexpansion and cloning.

Sporozoite Inhibition Assay. Sporozoite inhibition assayswere modified from Preston and Brown (23). Sporozoitesuspensions (0.25 ml) were prepared from infected ticks at aconcentration of 1 tick equivalent per ml in Eagle's minimalessential medium (GIBCO) and were incubated with an equalvolume of mAb (as either undiluted culture supernatant orascites diluted 1:100) or rabbit serum (diluted 1:4). After 1 hrat 37TC in 5% CO2 in air, 1 x 106 bovine peripheral bloodmononuclear cells, isolated from uninfected bovine blood(16), was added in 0.5 ml of RPMI medium with 16%heat-inactivated fetal calf serum. Each sporozoite:antibody

Abbreviation: mAb, monoclonal antibody(ies).tTo whom reprint requests should be addressed.§The sequence reported in this paper has been deposited in theEMBL/GenBank data base (accession no. M23460).

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combination was set up in quadruplicate in 1-cm3 wells of48-well plates (Costar) and cultures were incubated at 370C in5% CO2 in air.Four days later, cytocentrifuge smears were made from

each well and Giemsa stained. Two hundred cells werecounted from each to obtain the number of infected cells.Control incubations were performed at the same time usingstandard antibodies. The standard used for the mAb inhibi-tions was an anti-Taenia saginata mAb, whereas the stan-dards used for the rabbit antibodies were the preimmunerabbit sera.The results obtained were analyzed by the Mann-Whitney

U test for nonparametric data (24) and those showing a valueofP < 0.05 between the median percentage infection valuesfor standard and test antibodies are taken to be significant.Percent inhibition was calculated by comparing the medianpercentage infection for the standard antibody (X) with themedian percentage infection for the test antibody (Y) in theequation: [(X - Y)/X] x 100.

Immunoblotting and Sample Preparation. Samples forimmunoblotting were boiled for 5 min in Laemmli samplebuffer (25) and electrophoresed on 7-20% polyacrylamidegels containing 0.1% NaDodSO4. The separated proteinswere electrophoretically transferred to nitrocellulose sheets,which were then probed with the relevant antibodies asdescribed (26). The samples used were lysates of eithersporozoites or induced lysogens.

Sporozoites were purified on Percoll gradients fromground-up infected ticks (20). Harvested sporozoites werewashed once in phosphate-buffered saline (0.137M NaCl/2.6mM KCl/3.2 mM Na2HPO4/1.5 mM KH2PO4, pH 7.2), andthe pellets were resuspended in 0.5 ml of phosphate-bufferedsaline containing protease inhibitors (phenylmethylsulfonylfluoride, 1 mM; Na-p-tosyl-L-lysine chloromethyl ketone, 2.5mg/100 ml; N-tosyl-L-phenylalanine chloromethyl ketone, 5mg/100 ml) and stored at -70°C. Crude infected and unin-fected tick material was obtained directly from the superna-tant of a ground-up tick suspension before filtration (16).Purified uninfected tick material was harvested from a Percollgradient from the 1.07/1.09 specific gravity interface at whichsporozoites band. Between 1 and 3 tick equivalents ofsporozoites were used per gel track.Lysogens were grown overnight at 30°C. They were then

diluted 1:5 and grown for 2 hr at 30°C. Isopropyl 83-D-thiogalactopyranoside was added to 10 mM and the cellswere placed at 42°C for 30 min with occasional shaking.Incubation was continued for a further 60 min and theinduced lysogens were then harvested and lysed in Laemmlisampler buffer (25). The equivalent of 0.5 ml of inducedculture was loaded per gel track.

Construction and Screening of a Genomic Expression Li-brary. DNA was isolated by standard procedures (27) frompurified piroplasms of the Hissar stock. This was thenrandomly sheared and inserted into Agtll as described (28,29). The resulting library of 106 recombinants was plated onY1090 (hsdR-, M+) and screened with mAb 1A7, and posi-tive clones were located by using horseradish peroxidase-linked second antibody (30). Two positive clones (Agtll-SR1and -SR2) were selected and subjected to two further roundsof purification. Lysogens harboring these recombinants wereprepared in Y1089 as described (31) and then processed forimmunoblotting as outlined above. The DNA inserts fromthese two phage were released with restriction endonucleaseEcoRI and ligated into pUC18 for use as probes in subsequenthybridization analysis.

Preparation of a Rabbit Antiserum Against Fusion Proteins.A 30-ml culture of lysogen carrying Agtll-SR1 was inducedand the cells were harvested and prepared for NaDodSO4/polyacrylamide gel electrophoresis as described above. Thegel band containing the fusion protein was excised from a

preparative gel and electroeluted using a model 422 electro-eluter (Bio-Rad). After overnight dialysis against phosphate-buffered saline the fusion protein was used to immunize arabbit. Three injections, at monthly intervals, with -50 pug ofAgtll-SR1 fusion protein, were given intramuscularly. Thefirst two doses were combined with Freund's completeadjuvant and the third was combined with Freund's incom-plete adjuvant. Antiserum was taken 10 days after the thirddose. A control rabbit received adjuvant combined with anirrelevant bacterial antigen from Dermatophilus congolensis.DNA Sequence Analysis. Sequence determination was per-

formed by using the chain-termination method (32), aftersubcloning the EcoRI fragment of Agtll-SR1 into M13tgl31(33) in both orientations.

Nucleic Acid Blotting and Hybridization Analysis. GenomicDNA was extracted (27) from purified piroplasms. Threemicrograms was digested with restriction endonucleaseEcoRI as specified by the manufacturer (GIBCO-BRL). TheDNA was then subjected to electrophoresis through O.9oagarose gel (27), followed by blotting onto a nylon membrane(GeneScreenPlus, DuPont) by the method of Southern (34).The filter was prehybridized for 2 hr at 65TC in 0.5 M sodiumphosphate buffer (pH 7.5) with 7% NaDodSO4 and 100 ttg ofsalmon sperm DNA per ml and hybridized overnight in thesame solution containing the Agtll-SR1 insert, labeled with32p, by the random priming method (Polymeraid; P & SBiochemicals, Liverpool, U.K.), to a specific activity of 109

40

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so

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1 2 3 4 5 6 7 8

Irr- ThHoH

9 10 11 1213 14

FIG. 1. Inhibition of sporozoite entry into peripheral bloodmononuclear cells by antibodies against a surface epitope of Theileriaannulata sporozoites. The inhibition assays were performed asdescribed in the text. The data are presented in the form of two barcharts. The upper chart shows the median percentage of mononu-clear cells infected in the presence of the antibodies, whereas thelower chart shows the same data expressed as the percentageinhibition of infection. Each bar represents a different antibody usedin the test. Those bars marked with a star (*) show a significantdifference (P < 0.05) from the standard antibodies included ascontrols. Bars 1, control mAb against Taenia saginata (standardantibody); bars 2, mAb 1A7 (anti-sporozoite); bars 3, 5, and 7,control serum from a rabbit before it received Agtll-SR1 fusionprotein (standard antibody); bars 4, 6, and 8, rabbit antiserum againstAgtll-SR1 fusion protein; bars 9, 11, and 13, control rabbit serumbefore receiving Dermatophilus congolensis; bars 10, 12, and 14,control rabbit antiserum against Dermatophilus congolensis. Thesporozoites used were derived from three different stocks of theparasite: bars 1-4, 9, and 10, Theileria annulata (Ankara); bars 5, 6,11, and 12 Theileria annulata (Gharb); bars 7, 8, 13, and 14, Theileriaannulata (Hissar).

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cpm/,g. The hybridized filter was washed for 1 hr in 2x SSC(SSC = 0.15 M NaCl/15 mM sodium citrate) and 1 hr in 0.2xSSC at 650C and then subjected to overnight autoradiographyat -70'C with an intensifying screen.RNA was isolated from all stages by homogenization in 4

M guanidium thiocyanate with 50 mM Tris, pH 7.5/10 mMEDTA/1% 2-mercaptoethanol, followed by precipitationwith 0.75 vol ofethanol and phenol extraction ofthe resultingprecipitate. The RNA was isolated by overnight precipitationon ice in 3 M sodium acetate (pH 5.5). Poly(A)+ RNA wasprepared on oligo(dT)-cellulose (Boehringer), as described(27). The RNA was denatured, electrophoresed, and blottedonto nylon membranes (GeneScreenPlus, DuPont) as de-scribed by Gehring et al. (35). Probing was performed usingthe Agtll-SR1 insert prepared as described above.

RESULTS AND DISCUSSIONTwenty anti-sporozoite mAbs were screened for their abilityto block sporozoite invasion of mononuclear cells in an invitro assay similar to that used to demonstrate the blockingaction of hyperimmune bovine serum (23, 36). One mAb,1A7, blocked sporozoite invasion to a significant degree suchthat 66% of sporozoite infectivity was inhibited (Fig. 1). In anindirect fluorescent antibody test, mAb 1A7 reacts positivelywith formalin-fixed sporozoites from three geographicallydistinct stocks (data not shown). These stocks, from Ankara(Turkey), Hissar (India), and Gharb (Morocco), are known todiffer by isoenzyme analysis (37) and in their reactivity to apanel ofanti-macroschizont mAbs (38). Viable Ankara sporo-zoites are also labeled in the indirect immunofluorescenceassay, indicating that a surface antigen is recognized (not

shown). Other parasite stages-namely, the macroschizontand piroplasm-are not recognized by mAb 1A7.To identify the molecules carrying the epitope recognized

by mAb 1A7, we performed immunoblot analysis. By usinglysates of purified sporozoites from the same three stocksdescribed above, the results show that the epitope resides oneach member of a family of four protein doublets of Mrs85,000, 72,000, 63,000, and 54,000 (Fig. 2 Left, tracks 2, 3,and 6). Each stock shows a similar pattern of reactivity withmAb 1A7, the only observed difference being in the size ofthe Mr 85,000 doublet, which, in the Gharb stock, occurs atMr 80,000. Western blotting of crude homogenates frominfected ticks, containing all stages of sporozoite develop-ment, shows an extra band of higher molecular weight (Mr104,000) (Fig. 2 Left, track 4). Whether these multiple poly-peptides represent processing intermediates of a single geneproduct or whether each represents related but independentgene products remains to be determined.To extend this study and to obtain large quantities of these

molecules for immunization trials, we isolated parasite re-combinant DNA molecules encoding the mAb 1A7 epitope.This was achieved by inserting randomly sheared parasitegenomic DNA into the expression vector Agtll (29) andscreening the resulting library with mAb 1A7. Two phageclones, Agtll-SR1 and Agtll-SR2, expressing the mAb 1A7epitope were purified and the relationship between them isshown in Fig. 3 Upper. Lysogens harboring these two phagewere prepared and immunoblotting of induced lysogens re-vealed that each recombinant produces a specific ,B-galactosidase fusion protein reacting with mAb 1A7. Theirsizes appear to be 135,000 (Agtll-SR1) and 147,000 (Agtll-SR2) as shown in Fig. 2 Right (tracks 1 and 2), giving theilerial

Mol. wt.x 1000

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FIG. 2. Immunochemical characterization of the antigen defined by mAb 1A7. (Left) Immunoblots of proteins extracted from sporozoites.The antibodies used were mAb 1A7 (tracks 1-6), preimmune rabbit serum (track 7), and rabbit antiserum against fusion protein expressed byAgtll-SR1 (track 8). The tracks were loaded with the following extracts: crude uninfected ticks (track 1); Percoll purified sporozoites (Hissar)(track 2); Percoll purified sporozoites (Gharb) (track 3); crude infected ticks (Hissar) (track 4); Percoll purified uninfected tick material (track5); Percoll purified sporozoites (Ankara) (tracks 6-8). (Right) Immunoblot of proteins extracted from the induced lysogens of Agtll-SR1 (tracks1, 3, and 5-7) and Agtll-SR2 (tracks 2 and 4). The blots were probed with the following antibodies: mAb 1A7 (tracks 1, 2, and 5); mAb 4B11,a control anti-sporozoite mAb that recognizes different antigens (tracks 3 and 4); preimmune rabbit serum (track 6); antiserum against Agtll-SR1fusion protein (track 7).

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1 0.3

L I IX -

L

t ftr r r

1 1.3 1.70.1

GAA TTC CTA GTG AAG GAT GTA AGC GAA GGlu Phe Leu Val Lys Asp Val Ser Glu G

GGA ATA GGA GAT TTA AGT GAC CCG AAT A

Gly Ile Gly Asp Leu Ser Asp Pro Asn S

CCA AAT GCA AAA CCA GCC GAA CTT GGA CPro Asn Ala Lys ,Pro Ala Glu Leu Gly Pi

GTG ATA CAA AAT GTA CCA TCA GAC CCC TVal Ile Gin Asn Val Pro Ser Asp Pr~o S

ACA CCA ACA CAG CCT TCA AAT TTG CCA C

Thr Pro Thr Gin Pro Ser Asn Leu Pro G

ACA ACA GGG CCG GGG AAC GGG ACG GAT GThr Thr Gly Pro Gly Asn Gly Thr Asp G

ACA GGA CCA GGT GGA AAC GGG GAA GGA G

Thr Gly Prp Gly Gly Asn Gly Glu Gly G

TTG AAG GAA GGA GAA AAG AAA GAA GGA TLeLl Lys Glu Gly Glu Lys Lys Glu Gly L

AAG ATC AAA AAC AAA CTC TTG GGC TCA GLys Ile Lys Asn Lys Leu Leu Gly Ser G

GTC ACA AGG AAT TCVal Thr

FIG. 3. (Upper) Relationship between the inseAgtll-SR1 and Agtll-SR2 that express the mAb I

numbers refer to lengths in kilobases. The letter r 4restriction sites; L and R designate left and right, ihatched bar is the Agtll-SR1 insert sequence and in(occurs in Agtll-SR2. The horizontal arrow denotetranscription. (Lower) Nucleotide sequence of thAgtll-SR1 and the predicted amino acid sequence. Iis shown in phase with ,B-galactosidase. The twoindicated by a line above the sequence. The prounderlined to emphasize their high frequency.

peptide portions of Mr 19,000 and Mr 31,0(0When we isolated the DNA inserts from thephage it was noted that the insert in Agtll-Sfbase pairs (bp) in size (data not shown), wicoding capacity OfMr '11,000-12,000 only. 'SR1 fusion protein appears to include theilsize Mr 19,000, an anomaly exists that

investigate.To this end, we sequenced the Agtll-SR

Lower) and obtained the predicted aminowhich contains a protein coding region re

proline (10% overall with one stretch of 25%acids 20 and 63). Such a high proline contentbeen reported to slow migration on NaDodamide gels (39) and explains the anomalous siprotein.

To confirm that the clones genuinely encode and expressR a sporozoite epitope, a rabbit was immunized with the--Xgtll-SR kAgtll-SR1 fusion protein purified from NaDodSO4/poly-

acrylamide gels and anti-SR1 serum was produced (seeMaterials and Methods). This serum reacts strongly with theoriginal fusion protein in immunoblots (Fig. 2 Right, track 7)and, more importantly, with the same four protein doubletsas mAb 1A7 on sporozoite extracts (Fig. 2 Left, track 8).

R Significantly, the rabbit serum retains the functional ability of__ Xgt11-SR2 mAb 1A7 in strongly blocking sporozoite infectivity in vitro

such that up to 100% inhibition is achieved (Fig. 1). Thisneutralizing activity operates against all three sporozoitestocks and is not observed with preimmunization serum or

iAi CAG GTT with serum from a rabbit immunized with an irrelevantlu Gln Val bacterial (Dermatophilus congolensis) antigen.

To continue characterization of the gene encoding the mAbGC AGA ACA 1A7 epitope, Southern blot analysis was performed. Whener Arg Thr the Agtll-SR1 insert hybridizes to EcoRI-digested piroplasm

DNA from Ankara and Hissar stocks, three bands areCT TCA CTA revealed (Fig. 4 Left, tracks 1 and 2). Since there are noro Ser Leu internal EcoRI sites in this clone, we favor the interpretation

that there are three copies of the gene in both stocks. WeCA AAA GTG cannot formally exclude the possibility that there could be

two copies, with an EcoRI site polymorphism in the second~AA GTA CCA copy, giving rise to the three observed bands. Whicheverin Val Pro explanation is true, at present, we cannot distinguish whether

the three bands observed are due to the presence of two oriGA ACA ACA three gene copies per haploid genome or to alleles at a singleiy Thr Thr locus, since the parasite stocks used are not clones. It is

interesting to speculate that some of the four proteins present;GC AAA GAT on the mature sporozoites could be the result of expressioni1y Lys Asp of different genes.

To study the RNA transcripts encoding the mAb 1A7TA TTT CAA epitope, Northern blot analysis was performed on RNA.eu Phe Gln extracted from the salivary glands of infected ticks. Hybrid-

ization with the Agtll-SR1 insert revealed that a major RNA'GA TTC GAA species of 3.1 kilobases (kb) was specifically expressed in the

infected tick salivary gland (Fig. 4 Right). No hybridizationwas seen in the RNA from uninfected glands. Upon longerexposure, higher molecular weight bands of6.4 and 4.9 kb arevisible, which may represent precursors of the 3.1-kb se-

erts in the clones quence or may be independently transcribed in sporoblasts to1A7 epitope. The give rise to the Mr 104,000 antigen observed in Western blotsdesignates EcoRI of crude tick homogenates (Fig. 2 Left, track 4). The analysisrespectively. The was extended to RNA from other stages of the life cycle to

s the direction of see if expression is stage specific. Our results indicate (Fig.teinsert of clone 4 Right) that this gene is only expressed in the sporozoite andrhe reading frame presporozoite stages and thus affords a model system forEcoRI sites are studying the control of differential gene expression in this

line residues are important parasite.As there is no laboratory animal model for Theileria

annulata, work is necessary to immunize the bovine host0, respectively, with recombinant proteins derived from the gene we havese recombinant described, to see if protective immunity can be generated. ItU1 was only 330 is possible that anti-sporozoite antibody alone will not beSth a theoretical adequate to prevent infection as a few sporozoites maySince the Agtll- escape neutralization. However, it may serve to reduce theerial peptide of infective dose sufficiently to allow the development of awe went on to protective immune response to the macroshizont stage, for

which antigens on the surface of parasitized leukocytes,1 insert (Fig. 3 recognized by genetically restricted cytotoxic T lympho-acid sequence, cytes, are believed to be of major importance (40). Indeed,latively rich in the recombinant sporozoite surface antigen could be used asbetween amino part of a "cocktail" vaccine together with antigens from

t has previously other stages. Thus a number ofdifferent immune mechanismsIS04/polyacryl- would be activated to simulate the protective immune re-ize of the fusion sponses elicited during active infection with Theileria annu-

lata.

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kb

to -- 3.4

.........

2 3 4

kb

1 2

FIG. 4. Hybridization of the insert of the Agtll-SR1 clone to parasite nucleic acids. (Left) Southern blot analysis. EcoRI-digested piroplasmDNA (3 Ig per track) from Ankara (track 1) and Hissar (track 2) stocks of Theileria annulata, probed with the insert of Agtll-SR1. (Right)Northern blot analysis. Total RNA (tracks 1-4, 20 jug per track) and poly(A)+ RNA (tracks 5-9, 5 ug per track) were loaded as follows:uninfected, tick salivary glands (tracks 1, 3, and 5); tick salivary glands, infected with Ankara sporozoites (tracks 2, 4, and 6); an uninfectedbovine lymphosarcoma line, BL20 (30) (track 7); BL20 infected with Hissar macroschizonts (track 8); Hissar piroplasms (track 9). The blot wasprobed with the insert of Agtll-SR1.

We thank Richard Boid, Jane Glascodine, Mike Dyer, Geoff Hide,Alex Gray, Philip Mason, and Jane Kinnaird for helpful discussionand criticism; we are grateful to Cathy Harrison, Lesley Bell, GwenWilkie, and Mary Thomas for technical assistance. For art work wethank Bob Munro and Alan May and for typing the manuscript wethank Carol Law and Afshan Fairley. The anti-Taenia saginata mAbused as a control was kindly donated by Leslie Harrison and theanti-Dermatophilus congolensis antiserum was raised by Ian Heronand Alex Morrow. This work was supported by the OverseasDevelopment Administration and the Wellcome Trust. P.B. wassupported by a Royal Society European exchange fellowship fundedby the Deutsche Forschungsgemeinschaft.

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