characterisation of the 33 kda piroplasm surface antigen of theileria orientalis/sergenti/buffeli...

Veterinary Parasitology 104 (2002) 103–117

Characterisation of the 33 kDa piroplasm surfaceantigen of Theileria orientalis/sergenti/buffeli

isolates from West Java, Indonesia

Marc Govaerts a,1, Peter Verhaert b,2, Frans Jongejan c,d,Bruno M. Goddeeris a,∗

a Laboratory of Physiology and Immunology of Domestic Animals,Katholieke Universiteit Leuven, Leuven, Belgium

b Research Unit Comparative Animal Physiology and Morphology, Zoological Institute,Katholieke Universiteit Leuven, Leuven, Belgium

c Department of Parasitology and Tropical Veterinary Medicine, Faculty of Veterinary Medicine,Utrecht University, Utrecht, The Netherlands

d Department of Tropical Veterinary Diseases, Faculty of Veterinary Science,University of Pretoria, Pretoria, South Africa

Received 15 February 2001; received in revised form 28 September 2001; accepted 9 October 2001

Abstract

The immunodominant 33/35 kDa antigen of a Theileria isolate from West Java, Indonesia, wascharacterised and immuno-affinity purified by use of a monoclonal antibody, KUL-a4, and wasshown to be representative of the T. orientalis/sergenti/buffeli group. The aminoterminal sequenceof the purified 35 kDa peptide (20 residues) was determined by automated Edman degradation andfound to correspond to the predicted amino acid sequence of a prospective p33 gene previouslysequenced from the same isolate. The cleavage site of a putative signal peptide was identified andconforms the (−3, −1) rule for signal peptidases. The existence of dimeric and trimeric forms ofthe p33/35 antigen is hypothesised from Western blot profiles. KUL-a4 appeared specific for theT. orientalis/sergenti/buffeli group. It did not recognise in indirect fluorescence antibody test (IFAT),

∗ Corresponding author. Present address: Laboratory of Physiology and Immunology of Domestic Animals,Faculty of Agricultural and Applied Biological Sciences, Katholieke Universiteit Leuven, Kasteelpark Arenberg30, B-3001 Heverlee, Belgium. Tel.: +32-16-321-427; fax: +32-16-321-994.E-mail address: [email protected] (B.M. Goddeeris).

1 Present address: Department of Veterinary Science, Queen’s University Belfast, Belfast, United Kingdom.2 Present address: Target Discovery Unit, NV Organon, Oss, The Netherlands.

0304-4017/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved.PII: S0 3 04 -4017 (01 )00621 -5

104 M. Govaerts et al. / Veterinary Parasitology 104 (2002) 103–117

intraerythrocytic bodies of Anaplasma marginale or piroplasms and schizonts of T. mutans, T. parvaand T. annulata, whereas cattle antisera raised to these species showed cross-reactivity in IFAT. Ithowever, appeared weakly cross-reactive in Western blot and ELISA, with the 34 kDa piroplasmantigen of one T. annulata (Gharb) isolate. The present study indicates that the isolated antigenbelongs to the p33/34 antigen family described within the T. sergenti/orientalis/buffeli group, anddocuments the group-specificity of one of its epitopes. © 2002 Elsevier Science B.V. All rightsreserved.

Keywords: Oriental theileriosis; p33 major piroplasm surface antigen; Signal peptide; Theileria buffeli; Theileriasergenti; Theileria orientalis

1. Introduction

Oriental theileriosis is a haemoparasitic disease of cattle due to apicomplexan protozoaof the Theileria orientalis/sergenti/buffeli group that are transmitted by ticks of the genusHaemaphysalis (Brown et al., 1990). The parasites are distributed worldwide, showingvarious degrees of pathogenicity, and have been either referred to as T. sergenti (Yakimoffand Dekhtereff, 1930), T. buffeli (Neveu-Lemaire, 1912, pp. 288–291), or T. orientalis(Yakimoff and Soudatschenkoff, 1931). No consensus has been reached so far to con-sider this group as a single species, to be historically named T. buffeli (Gubbels et al.,2000).

Theileriosis has been reported in Indonesian cattle and buffaloes since 1897 (De Blieckand Kaligis, 1912). Based on the distribution range of T. annulata excluding South East Asia(Dolan, 1992), and on the benign character of field and experimental cases, infections wereoriginally ascribed to T. mutans (Soekardono, 1989), and later to T. orientalis upon assayingthe serological cross-reactivity of field isolates with anti-T. orientalis antisera (Astyawati,1987). Except for the recent sequencing of the small subunit ribosomal RNA gene from anisolate of Medan, Sumatra (GenBankTM accession number AB000274), Indonesian para-sites have not been further characterised at the biological, serological or molecular level.Besides Giemsa-stained blood smear surveys, there is no serological information about itsprevalence in the various biotopes of this country (Siswansyah, 1990).

A number of serological tests has been described for the identification of T. sergenti,based on an indirect fluorescence antibody test (IFAT) (Fujinaga and Minami, 1981) andELISA (Shimizu et al., 1988). DNA probes and polymerase chain reaction (PCR) have alsobeen used for its molecular differentiation from two Australian T. buffeli strains (Kawazuet al., 1995).

In the present study an Indonesian isolate of T. orientalis/sergenti/buffeli wascharacterised by its 33 kDa antigen, a polymorphic piroplasm surface protein of theTheileria genus. Utilising a monoclonal antibody (mAb), the protein was identified, puri-fied and partly sequenced. The mAb was specific for the T. orientalis/sergenti/buffeli group and enables the development of serological assays specific for this group,for use in diagnostic and epidemiological studies in areas infested with different Theileriaspecies.

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2. Materials and methods

2.1. Parasites

2.1.1. Field isolatesThe two Theileria isolates used in this study both originated from Ongole zebu cattle of a

buffalo/Ongole mixed herd reared under free-grazing conditions on the hills of Jonggol, WestJava Province, Indonesia. The Jonggol-1 isolate originated from peripheral blood of a singlecow, while the Jonggol-2 isolate was a mixed isolate consisting of a pool of 20 ml-aliquots ofblood from two cows. The Jonggol-1 isolate was expanded by three serial blood passagesin haemoprotozoan-free splenectomised Bos taurus calves, at day post inoculation (dpi)14 and 52, respectively, whereas Jonggol-2 was maintained in a single splenectomisedcalf. Jonggol-1 cryopreserved stabilates were prepared and appeared free of non-Theileriaspp. parasites upon in vivo suboculation. All animals were kept in a tick-free area.

2.1.2. Reference isolatesAntigens from A. marginale and piroplasms, and schizonts of various Theileria spp. were

used for antibody assays as listed in Table 1.

2.2. Antibodies

2.2.1. Antisera against Jonggol isolatesJonggol-1 was passaged a fourth time into a non-splenectomised calf at dpi 81 for the

production of positive control antiserum. Sera were collected from BalbC mice immu-nised three times at 3-week intervals with piroplasms of Jonggol-2, 10 days after the lastimmunisation.

2.2.2. Production of mAbsThe Jonggol-2 mixed isolate was selected to produce mAbs with the broadest

specificity. Infected erythrocytes were collected 40 dpi at a 17% parasitaemia and free

Table 1Reference parasite stocks

Species Stock/isolate Antigen source Area of origin

T. buffeli Wacol Purified piroplasms Queensland, AustraliaT. mutans Intona Stabilated piroplasms Transmara, KenyaT. parva – Fresh piroplasms KenyaT. parva Muguga Fresh schizonts Kikuyu, Kenya

Katete, Zam-2, -3,-5, -22, -23

Fresh schizonts Zambia

T. annulata Hissar Purified piroplasms TurkeyGharb Purified piroplasms fixed piroplasms MaroccoAnkara Fixed schizonts Turkey

A. marginale Jonggol Fixed intraerythrocytic bodies Indonesia


piroplasms were prepared by NH4Cl lysis. The piroplasms were washed by centrifugation(Katende et al., 1990) and lysed on ice by a 15 min sonication (1/8 in. tapered microtipprobe, relative output 24%; 600 W VCX600 sonicator, Sonics and Materials, Danbury,CT, USA) in phosphate-buffered saline glucose (PSG), 5 mM EDTA, 1 mM phenylmethyl-sulfonylfluoride (PMSF). Six-week old BalbC mice were immunised intraperitoneally(i.p.) and hybridomas were produced using standard procedures (Harlow and Lane, 1988).Positive hybridoma cell lines were selected in IFAT and subcloned by limiting dilution with50 IU recombinant mouse interleukin-6 (Boehringer Mannheim, Mannheim, Germany) permillilitre of culture medium (Bazin and Lemieux, 1989).

Hybridoma supernatants of single- and double-colony wells were screened in IFAT onJonggol-2-infected blood smears 10 days after fusion. Twenty-one positive wells wereidentified and subcloned thrice. Clone KUL-a4 (IgG1 isotype) was selected for furtherstudy based on its staining characteristics in Western blot of the parasite. Ascitic fluid wasproduced and the mAb was purified on a protein G column.

2.2.3. Reference sera and mAbsBovine antisera and mAbs raised against different Theileria species, that were assayed

for cross-reactivity, are listed in Table 2. A panel of 19 pre- and post-inoculation bovineanti-tick borne disease antisera, listed in Table 3, was similarly tested.

2.3. Antibody tests

2.3.1. IFATJonggol-1 and 2 acetone-fixed blood smears were prepared for IFAT at passage 3, dpi

64 and dpi 419, respectively. IFAT was carried out on Jonggol-1 and 2, T. parva, T. mutans(Intona), and T. annulata (Gharb) piroplasms, as described previously (Burridge, 1971), andon T. parva (Muguga, Katete, Zambia 2, 3, 5, 22, 23) and T. annulata (Ankara) schizonts(Goddeeris et al., 1982). Using as secondary antibody, either a rabbit anti-bovine IgG (wholemolecule) FITC conjugate (Sigma, St. Louis, USA) for cattle sera or a sheep anti-mouseIgG F(ab′)2 FITC conjugate (Sigma) for mouse antibodies, the positivity cut-off value wasset at a serum dilution of 1:100 on all antigens, based on pre-infection sera results.

2.3.2. Antibody-detection ELISAIn brief, flat-bottomed microplates were coated with 1 ug/well of protein G-purified mAb

KUL-a4, blocked, and incubated with a sonicate of Jonggol-1 infected erythrocytes collected

Table 2Reference bovine antisera and mAbs

Specificity Stock Origin

T. annulata Gharb + Ankara Marocco + TurkeyT. parva Marikebuni Coast Province, KenyaT. buffeli Wacol Queensland, AustraliaNegative control serum – BelgiumT. mutans native p32 mAbs TmIII.C2 and Tm1H1.A10T. mutans recombinant p32 mAbs 4C11.B7 and 4G2.G5


Table 3IFAT titres of bovine anti-tick borne diseases antisera on Jonggol-2 piroplasms

Antiserum IFAT titre on

Specificity Stock dpi Jonggol-2 piroplasms Homologous antigen

T. sergenti Jeju, Korea 43 6400 5120T. sergenti Fukushima, Japan 42 6400 5120T. buffeli – 66 6400 5120T. buffeli – 28 25600 10240T. orientalis Texas 96 6400 2560T. orientalis Essex, UK 74 6400 1280

T. velifera – 43 100 2560T. taurotragi Idobogo 59 400 1280T. taurotragi Chilington 34 <100 1280T. parva Pugu 2 42 400 2560T. mutans – 49 100 1280T. annulata Ankara 22 400 NAa

T. annulata Bahrein 73 1600 2560

A. marginale – 21 <100 NAA. marginale Texas 22 100 NAA. centrale Korea 52 <100 NA

B. bovis – 28 100 NAB. bigemina – 16 100 NAB. bigemina Nigeria 19 400 NA

a Non-available.

at passage 3, dpi 90 and diluted in PBS, 0.1% casein, 0.05% Tween 20, in order to capture thenative p33 antigen. After washing, sera diluted 1:100 were applied to the wells and followinga second wash, wells were incubated with an anti-bovine IgG peroxidase conjugate (Sigma).Antibody binding was quantified by an H2O2/ABTS substrate/chromogen 1 h incubation,and optical densities read at 450 nm.

2.3.3. SDS-PAGE and Western blotJonggol-2 piroplasms were prepared at dpi 468 from blood at a 19% parasitaemia by

sonication (1/2 in. probe, relative output 40%) under fluorescent acridine orange stainingmonitoring (Trees, 1974), and purified on a Percoll® 1.040/1.050 density step gradient(Walker and McKellar, 1983). Samples of 10 �l containing 3 �g or 3 × 106 solubilisedJonggol-2 piroplasms were electrophorised on a sodium dodecylsulfate polyacrylamide gel(SDS-PAGE).

Jonggol-1 piroplasms were collected at passage 3, dpi 97, 23% parasitaemia, andprepared by blood sonication without density gradient purification. They were washedand solubilised to yield 10 �g protein or 107 Jonggol-1 piroplasms per 14 �l SDS-PAGEsample.

Purified piroplasms of T. annulata Hissar and Gharb stocks, as well as of the Queenslandisolate of T. buffeli, were lysed as above, yielding 14 �l SDS-PAGE samples estimated at10 �g or 107 piroplasms.


Antigen samples were loaded on a 12% SDS-polyacrylamide gel after denaturation at99 ◦C for 3 min, either under non-reducing or reducing conditions (2-mercaptoethanol:89 mM), and electro-blotted onto a 0.45 �m nitrocellulose membrane (Biorad, Hercules,USA) (Towbin et al., 1979). Blots were developed using the biotin/avidin peroxidase system(Vectastain ABC Reagent Kit®, Vector Laboratories, Burlingame, USA).

Deglycosylation was based on Schiff’s periodic acid (PAS) treatment of the antigen, eitherbefore electrophoresis (Zhuang et al., 1993) or after blotting (Woodward et al., 1985).

2.4. P33 antigen purification and sequencing

Protein G-purified mAb KUL-a4 was coupled to cyanogen bromide-activated Sepharose4B beads (Pharmacia Biotech, Uppsala, Sweden) using the manufacturer’s instructions, andcoupled beads were packed into a 2 ml column.

The Jonggol-1 isolate was selected for antigen purification, both to minimise the presenceof antigen variants in mixed isolates (Gubbels et al., 2000), and directly align the peptidesequence with the p33 gene sequence identified in that isolate (GenBankTM AF102500;Govaerts et al., 1998). A pellet of 5 × 109 purified Jonggol-1 passage 3, dpi 97 piroplasms,was lysed for 3 h at 4 ◦C in 2 ml of binding buffer (Tris–HCl 50 mM, pH 8.0, NaCl 0.5 M)supplemented with EDTA 5 mM, PMSF 1 mM and 0.5% Nonidet NP40, and soluble anti-gens were passed three times over the column, which was subsequently washed with bindingbuffer. A total of 150 �g of purified antigen was eluted with a glycine–HCl 50 mM, NaCl0.5 M, pH 2.7 buffer.

Twenty-five micrograms of purified antigen, dialysed against deionised MilliQ® waterand reconcentrated to 20 �l, was diluted in a sucrose buffer (sucrose 0.1 M, SDS 3%, Tris62.5 mM, pH 6.9, Na2EDTA 2 mM, 2-mercaptoethanol 1% (v/v) and Bromophenol Blue0.05% (w/v)) and incubated at 37 ◦C for 15 min prior to loading on an SDS-PAGE gel.Electrophoresis was carried out adding 0.1 mM thioglycollate to the running buffer, andsamples were electro-blotted onto a polyvinylidene difluoride (PVDF) membrane (Biorad)following the manufacturer’s protocol, using a CAPS pH 11 blotting buffer to improveblotting (Legendre et al., 1993).

Bands were visualised on the membrane by Coomassie Brilliant Blue (CBB R-2500.025%, methanol 40%). Relevant bands were cut out and aminoterminal sequencing wasperformed in a Beckman LF3600TC automated sequencer (Beckman, Fullertone, USA),using the Edman phenylisothiocyanate degradation cycle principle (Findlay and Geisow,1989).

Peptide sequences were analysed using the PSORT II (Horton and Nakai, 1997) andSignalP v2.0 (Nielsen et al., 1999) software packages.

3. Results

3.1. Characterisation of Jonggol isolates

Anti-T. sergenti, -T. buffeli and -T. orientalis sera did fully cross-react (similar titresas homologous titres) with Jonggol-2 piroplasms (Table 3). Anti-Jonggol-1 bovine sera,

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Table 5Cross-reactivity of anti-T. annulata antisera with the Jonggol-1 native p33 antigen in ELISA (optical densities(OD) are expressed as test sample OD − blank well OD)

Antiserum OD

Specificity Stock dpi

T. orientalis/sergenti/buffeli Jonggol-1 55 0.749T. annulata Gharb + Ankara 226 0.166T. annulata Ankara 22 −0.012T. annulata Bahrein 73 0.004

as well as bovine antisera raised against T. annulata (Ankara, Bahrein, Gharb + Ankara),T. parva (Marikebuni, Pugu2), T. taurotragi (Idobogo) and B. bigemina (Nigeria) showedclear reactivity in IFAT with piroplasms or schizonts of Jonggol-2, T. annulata (Gharb,Ankara) and T. parva (Muguga, Katete, Zambia) (Tables 3 and 4). Bovine sera againstT. mutans, A. marginale (Texas), B. bovis and another B. bigemina isolate showed a weakcross-reactivity with Jonggol-2 piroplasms in IFAT (Table 3). Another anti-A. marginaleserum and anti-A. centrale (Korea) and -T. taurotragi (Chilington) sera were not cross-reactive. The two anti-T. mutans recombinant p32 mAbs did not react with the Jonggol-2piroplasms in IFAT nor in ELISA, neither did the anti-T. mutans native p32 mAbs in ELISA.The Jonggol-1 native p33 antigen captured by KUL-a4 appeared slightly cross-reactive inELISA with anti-T. annulata (Gharb + Ankara) sera but not with anti-T. annulata (Ankara)or (Bahrein) sera (Table 5).

3.2. Production of a Jonggol-specific mAb

KUL-a4 specifically stained the intraerythrocytic piroplasms of both Jonggol-1 and -2isolates in IFAT. No cross-reaction was observed in IFAT with A. marginale (Jonggol)bodies, T. annulata (Gharb), T. mutans (Intona) and T. parva piroplasms, nor with schizontsof T. annulata (Ankara) and T. parva (Muguga, Katete, and Zambia 2, 3, 5, 22, 23).

3.3. Western blot analysis

When probed with anti-Jonggol-1 positive control serum, the Jonggol-1 lysate displayeda strong double band with a molecular mass of 35/33 kDa, weak bands at 71/69 and105/97 kDa, and faint bands at 29, 24 and 23 kDa (Fig. 1A, lane 2). However, 35 and 29 kDabands were also observed when probed with negative control serum (Fig. 1A, lane 3).

When probed with homologous bovine antisera, a triple 34/33/31 kDa band was observedon T. buffeli (Wacol) piroplasms (Fig. 2A, lanes 2 and 4), and major bands at 36, 33, 21 and20 kDa on T. annulata (Gharb and Hissar) piroplasms (Fig. 2A, lanes 1 and 3).

KUL-a4 identified a double band with a molecular mass of 32.8 and 34.8 kDa (±1.2 kDa),further called 33 and 35 kDa bands, on Jonggol-1 and -2 lysates (Fig. 1A, lane 1, and Fig. 1B,lanes 1 and 2). Lysates prepared in non-reducing conditions displayed the same bands asthose prepared under reducing conditions, thus revealing a monomeric molecule. Whenprobed with sera from immunised mice to comparatively assess the immunodominance of

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Fig. 2. Western blots of T. annulata (Gharb, Hissar) and T. buffeli (Wacol) piroplasm lysates probed underreducing conditions with: (A) Homologous antisera 1:100: (1) T. annulata (Hissar) probed with anti-T. annu-lata (Gharb + Ankara) antiserum, (2) and (4) T. buffeli (Wacol) probed with anti-T. buffeli (Wacol) antiserum, (3)T. annulata (Gharb) probed with anti-T. annulata (Gharb + Ankara) antiserum. (B) KUL-a4 ascitic fluid 1:100:(1) T. annulata (Hissar), (2) T. annulata (Gharb), (3) T. buffeli (Wacol).

the 33/35 kDa antigen, one extra antigen band at 25 kDa dominated the blot, but only undernon-reducing conditions (Fig. 1B, lanes 3 and 4).

A shift in the intensity of detection of the antigen bands, from the 35 to the 33 kDaband, was observed throughout the storage of the antigen at 4 ◦C. The 35 kDa band waspredominantly stained upon antigen preparation (Fig. 1C, lane 1), whereas both bandsstained equally intense after 8 days of antigen storage (Fig. 1B, lanes 1 and 2) and only the33 kDa band could be detected from a 9-week old antigen preparation (Fig. 1C, lane 2).

No antigens could be detected by KUL-a4 on Western blots of T. annulata (Hissar)piroplasms (Fig. 2B, lane 1). Only very faint bands were recognised at 34, 32 and 31 kDaon the T. annulata (Gharb) antigen and a double band was stained at 34/33 kDa on theT. buffeli (Wacol) antigen (Fig. 2B, lanes 2 and 3).

3.4. Glycosylation of the Jonggol-2 33/35 kDa antigen

Deglycosylation by periodate treatment of the antigen yielded conflicting results on thenature of the antigen. Treatment of the antigen after blotting, failed to demonstrate anyglycosylation of the epitope recognised by the mAb, since the two bands remained visibleat 33 and 35 kDa, both on strips of non-reduced or 2-mercaptoethanol-reduced antigen.

PAS treatment of the antigen lysate prior to electrophoresis induced no change in thedetection of antigen by KUL-a4, when carried out under non-reducing conditions (Fig. 1D,lanes 3 and 4). However, when carried out on reduced antigen lysates, it resulted in the totaldisappearance of the antigen bands (Fig. 1D, lanes 1 and 2).


Fig. 3. SDS-PAGE (A) and Western blot (B) of Jonggol-1. (A) SDS-PAGE of: (1) 10 �g piroplasm lysate, (2)1.28 �g purified 33/35 kDa antigen. (B) Western blot of the purified 33/35 kDa antigen (1.28 �g/lane) probedwith: (1) KUL-a4 hybridoma supernatant, (2) anti-Jonggol-1 antiserum 1:100, (3) negative control serum 1:100,(4) anti-T. buffeli (Wacol) antiserum 1:100.

3.5. Aminoterminal sequencing of the 35 kDa polypeptide

The immunoaffinity-purified Jonggol-1 antigen displayed a mass of 35 kDa in SDS-PAGE(Fig. 3A, lane 2) and of 35/33 kDa in Western blots as evidenced by KUL-a4 (Fig. 3B, lane 1).Probing with bovine anti-Jonggol-1 and anti-T. buffeli (Wacol) sera revealed only the 35and 33 kDa bands (Fig. 3B, lanes 2 and 4, respectively), whereas no bands were detectedwith the negative control serum (Fig. 3B, lane 3). The 35 kDa band was aminoterminallysequenced for the first 20 residues as “AEEKK DAKAE EKKDL TLEVN”.

4. Discussion

Two Theileria spp. isolates (Jonggol-1 and -2) were collected in West Java, Indonesia,and their position within the Theileria genus confirmed by characterising and sequenc-ing their piroplasmic 33/35 kDa antigen. To this purpose a mAb specific for that antigenwas produced and used for antigen identification in Western blot and purification by im-munoaffinity chromatography. The 32.8 and 34.8 kDa bands identified in Western blotsof both Jonggol-1 and -2 isolates by mAb KUL-a4, matched the 32 and 34 kDa proteinbands described for T. buffeli (Kawazu et al., 1991a), the piroplasmic p33 or p34 proteinspreviously described for T. sergenti (Kawazu et al., 1992a) and the p32 piroplasm surfaceantigen family in Theileria parasites (Shiels et al., 1995). The 20 first residues obtained byautomated Edman degradation in Jonggol-1, fully correspond to the N-terminus (residues23–42) of the nascent peptide, predicted from the p33 gene identified in the same isolate, as


CLGYFLIVSA20TAAEEKKDAK30AEEKKDLTLE40VNATQAEHVT50 (GenBankTM

AF102500, Govaerts et al., 1998). The purified antigen is thus encoded by the gene wepreviously identified and sequenced. Based on Western blot and sequence homology for the33/35 kDa antigen, the Jonggol isolates appear to belong to the T. orientalis/sergenti/buffeligroup. Notwithstanding this serological cross-reactivity and their common KUL-a4 epitope,one cannot be 100% sure that the two isolates are identical.

The 35 kDa antigen sequence-based positioning of Jonggol-1 and -2 isolates withinthe T. orientalis/sergenti/buffeli group was corroborated by the highest cross-reactivitiesexpressed by antisera raised to this group, on Jonggol-2 piroplasms in IFAT, and thelower reactivity of anti-Jonggol-1 antiserum on antigens of other taxonomic groups. Thenon-recognition of Jonggol-2 piroplasms by the anti-T. mutans mAbs was confirmed inELISA, both with the anti-recombinant, and -native, T. mutans p32 mAbs. The taxonomicposition of both isolates is supported by the strong cross-reactivity observed in Western blots,of KUL-a4 with the T. buffeli (Wacol) 34/33 kDa antigen on one hand, and of the Australiananti-T. buffeli bovine sera with the Jonggol-1 purified p35/33 antigen, on the other hand.

Variant forms of the p33 antigen with molecular masses of 33, 34 and 35 kDa, havebeen documented for T. orientalis/sergenti/buffeli isolates (Kawazu et al., 1991b, 1992a,b;Zhuang et al., 1995; Gubbels et al., 2000). Due to increased and concomitantly decreaseddetection of the 33 and 35 kDa bands, respectively, it is likely that the band shift resultedfrom the degradation of the 35 kDa antigen into a 33 kDa product upon storage.

When probed with anti-Jonggol-1 bovine serum, Western blots of Jonggol-1 lysatesshowed, besides the strong double band at 35/33 kDa, weak doublets at 105/97 and 71/69 kDa(Fig. 1A, lane 2). These may correspond to dimeric and trimeric forms of the 35/33 kDaantigen (prospective Mr 70/66 and 105/99, respectively). These bands were not detectedby KUL-a4 on blots of total lysate, nor by Jonggol-1 bovine antiserum on purified antigen,which might indicate that the KUL-a4 epitope is hidden or modified in multimeric forms.The 35 kDa band detected by the negative bovine control serum on total lysates, appearsnon-Theileria-specific as it was absent in blots of purified p33/35 antigen, probed with thesame negative serum.

PAS-treatment of the Jonggol-2 antigen after blotting failed to demonstrate any N-glyco-sylation of the p33/35 antigen within the KUL-a4-recognised epitope. However, PAS-treatment of the antigen before electrophoresis indicated glycosylation of the p33 antigen,by rendering the epitope unaccessible, but only under reducing conditions. This mightreflect, on one hand, a possible inefficacy of the PAS N-deglycosylation of soluble anti-gens, if carried out under non-reducing conditions, and on the other hand, a possible inabilityto modify the KUL-a4 epitope on a membrane-bound antigen, due to cross-linking of theantigen onto the membrane or to a lack of access to the N-glycosylated groups.

A weak cross-reaction was observed in Western blots between KUL-a4 and a 34 kDaantigen in T. annulata piroplasms of the Gharb but not the Hissar stock. The p33 anti-gen of T. orientalis/sergenti/buffeli Jonggol-1 and -2 may thus share a cross-reactive epi-tope with a 34 kDa piroplasm antigen in certain strains of T. annulata with a reducedKUL-a4 affinity, since undetectable in IFAT. This is corroborated in ELISA by the slightcross-reactivity of anti-[Gharb + Ankara] sera on the Jonggol-1 p33 native antigen andthe absence of cross-reactivity of anti-T. annulata Ankara or Bahrein sera. Notwithstand-ing this cross-reactivity, KUL-a4 may be sufficiently specific for an oriental theileriosis


diagnostic ELISA for field use in Indonesia and other areas where T. annulata infectionsare not reported.

The gene encoding the p33 antigen in T. sergenti and T. buffeli has now been sequencedfor more than 25 isolates of this group (Kim et al., 1998). The prospective p33 genefragment we sequenced from the Jonggol-1 isolate (Govaerts et al., 1998) showed 88%homology with the p33 gene sequence of the Ikeda stock of T. sergenti (Kawazu et al.,1992b). The 100% homology between the 20-residues peptide fragment sequenced, andthe N-terminus predicted from the Jonggol-1 p33 gene sequence (Govaerts et al., 1998),is the first known formal demonstration directly linking the 33/35 kDa antigen detectedin a T. sergenti/orientalis/buffeli isolate, to its p33 gene. Based on the reading frame ofthe Jonggol-1 p33 gene, the N-terminal sequence we obtained from the native peptideindicates that it is produced by cleavage of a signal sequence from the nascent peptide.Analysis of the full-length p33 gene transcript revealed a 18-residues hydrophobic domainat the C-terminus, and a putative 22–23-residues cleavable signal sequence with positive,hydrophobic and polar domains (Von Heijne, 1990), at the N-terminus. This structure ischaracteristic of type Ia integral membrane surface peptides (Singer, 1990), and supportedby the N-terminal sequencing by Baek et al. (1994) of a Korean T. sergenti (Chonju) p33native antigen, which yielded the same AEEKK mature N-terminus.

The p33 signal peptide cleavage site was initially located prospectively on the second Glu24 residue of T. buffeli (Warwick) and Glu 25 of T. sergenti (Ikeda) (Kawazu et al., 1992b),yielding an EKKE mature peptide N-terminus. In view of the gene sequence, we obtainedpreviously (Govaerts et al., 1998), our data indicate that the signal peptidases involved inprocessing T. orientalis/sergenti/buffeli p33 antigens actually cleave the nascent peptidetwo residues upstream, at the Ala 22–Ala 23 bond in Jonggol-1 and T. sergenti (Ikeda).Our data are in agreement with to the so-called (−3, −1) rule for bacterial signal pepti-dase I (Von Heijne, 1990), defining Ala-X-Ala as their most common [−3 to −1] cleavagesite motif (Dev and Ray, 1990). This ATA ↓ AEEK cleavage site was predicted by thePSORT weight-matrix method for eukaryotic signals, and by the SignalP neural networkalgorithm and hidden Markov model methods when trained on Gram-negative bacteria datasets, but not on eukaryotic data sets. The eukaryotic set being dominated by mammaliansequences (Nielsen et al., 1999), apicomplexan signal peptidases may thus be more related,in their cleavage patterns, to bacterial leader peptidases than to mammalian signal peptidasecomplexes.

In conclusion, the present characterisation of the p33 antigen confirms the position of anIndonesian Theileria isolate within the T. sergenti/orientalis/buffeli group, with its closesthomology to the T. sergenti Ikeda stock of Japan. The availability of the mAb KUL-a4will enable the development of a native p33 antigen-based, antibody detection ELISA.Preliminary use of this mAb has already shown that such an ELISA is sufficiently specificfor field-use in Indonesia.

Acknowledgements

We are grateful to T. Jones and E. Kirvar, Centre for Tropical Veterinary Medicine,University of Edinburgh, for fruitful discussions and access to materials; to L. Parede,


Research Institute for Veterinary Science, Bogor, Indonesia, for kindly supplying us asp2/0-Ag14 myeloma cell line; to J. Mast, Katholieke Universiteit Leuven, for his assis-tance in Western blot assays; to A.J. Musoke, S.P. Morzaria, and J. Katende, InternationalLivestock Research Institute, Nairobi, to S. Geerts and D. Geysen, Prince Leopold Instituteof Tropical Medicine, Antwerp, and to W. Jorgensen, Queensland Department of PrimaryIndustries, for supply of samples; and to S.H. Siggit and E.H. Siregar, Bogor AgriculturalUniversity, for their support in Indonesia.

This study was carried out within the framework of the Indonesia–Belgium project“Parasitic and reproductive constraints on livestock production in Indonesia” between theKatholieke Universiteit Leuven, Belgium, and Bogor Agricultural University, Indonesia,under support of the Flemish Interuniversity Council and the Belgian Administration forInternational Cooperation. P. Verhaert was supported by the Flanders Fund for ScientificResearch.

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characterisation of the 33 kda piroplasm surface antigen of theileria orientalis/sergenti/buffeli...

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