reciprocal cross-protection induced by sporozoite antigens spag-1 from theileria annulata and p67...

Reciprocal cross-protection induced by sporozoite antigens SPAG-1

from Theileria annulata and p67 from Theileria parva

ROGER HALL1, NICOLA R.BOULTER1, C.G.DUNCAN BROWN2, GWEN WILKIE2, EROL KIRVAR2, VISH NENE3,

ANTHONY J.MUSOKE3, ELIZABETH J.GLASS4 & SUBHASH P. MORZARIA3

1Department of Biology, University of York, PO Box 373, York, Y010 5YW, UK, 2Centre for Tropical Veterinary Medicine, University of

Edinburgh, Easter Bush, Roslin, Midlothian, EH25 9RG, Scotland, UK, 3International Livestock Research Institute (ILRI), PO Box 30709,

Nairobi, Kenya and 4Roslin Institute, Roslin, Midlothian, EH25 9PS, Scotland, UK

SUMMARY

Theileria annulata and Theileria parva both possess a major

surface antigen on the sporozoite stage of the life-cycle,

called SPAG-1 and p67, respectively. In each case, these

antigens are vaccine candidates and have been shown to

induce a degree of homologous protection in earlier work.

These antigens share sequence homology and are serologi-

cally cross-reactive. Here, we con®rm that these antigens

confer protection against homologous species challenge.

More importantly, they mutually confer a degree of cross-

species protection raising the prospect of a common vaccine

in the future.

Keywords Theileria annulata, Theileria parva, sporozoite

antigen, vaccine, cross-protection

INTRODUCTION

Theilerioses impose a severe constraint on the productivity

of domestic ruminants. The cattle industry is under parti-

cular threat from two theilerial species. Theileria annulata,

which causes tropical theileriosis, is the most widespread

affecting large parts of Asia, northern Africa and southern

Europe. Theileria parva is the most virulent and affects

cattle in large parts of eastern, southern and central Africa

(Dolan 1989). Both these infections are partially controlled

by live vaccines. In the case of T. annulata, an attenuated

schizont vaccine is used, which is highly effective (Tait &

Hall 1990, Somerville et al. 1998). For T. parva, the

`infection and treatment' method, involving simultaneous

administration of a quanti®ed dose of sporozoite stabilate

and a long acting oxytetracycline is applied (Radley 1981).

Both vaccines carry risks, which include reduced ef®cacy

due to breakdown of the cold chain, disease due to immu-

nization and the possibility of introducing other contaminat-

ing pathogens due to inadequate quality control during

preparation. Also in the case of T. annulata, there is the

possibility of reversion to virulence. The T. parva vaccines

are mixtures or `cocktails' of different local strains which,

due to antigenic polymorphism, individually do not cross-

protect (Irvin & Morrison 1987). There are reports of

vaccine mediated introductions of novel virulent variants

of T. parva in certain areas (Mutugi et al. 1991). Finally,

logistical problems of manufacture, storage, stability and

particularly delivery, requiring a cold chain, all contribute to

the conclusion that the creation of a de®ned, cross-protec-

tive subunit vaccine effective against both species would be

an ideal solution.

Subunit vaccine research in Theileria has been focused to

date on surface antigens of the sporozoite stage of the life-

cycle. The antigens studied are called SPAG-1 and p67 from

T. annulata and T. parva, respectively. Both antigens were

originally de®ned by neutralizing monoclonal antibodies,

Parasite Immunology, 2000: 22: 223±230

q 2000 Blackwell Science Ltd 223

Correspondence: R.Hall

Received: 20 July 1999

Accepted for publication: 27 January 2000

which importantly recognized nonvariant epitopes within

each species (Williamson et al. 1989, Musoke et al. 1992).

This evidence of a common protective epitope is particu-

larly important in the case of T. parva which, as mentioned,

is quite heterogeneous in terms of cross-immunity pro®les,

and is thought to be due to polymorphism in the schizont

antigens targeted by cytotoxic T cells. Both SPAG-1 and

p67 antigens have been tested in a number of trials (Boulter

& Hall 1999) using a variety of regimes and both offer some

protection (Musoke et al. 1992, Boulter et al. 1994, Boulter

et al. 1995, Nene et al. 1996, Boulter et al., 1998, Gentschev

et al. 1998, Heussler et al. 1998, Honda et al. 1998). In the

case of p67 and T. parva, approximately 70% of the

immunized animals are protected to an LD70 challenge

with sporozoites inoculated by syringe and derived from

homologous and immunologically heterologous stocks of

the parasite (Musoke et al. 1993). Examination of the

predicted amino acid sequences of p67 and SPAG-1 reveals

that they are functional homologues being approximately

50% identical at the N- and C-terminal sections (Katzer et

al. 1994). Perhaps more importantly, they have been shown

to share epitopes both by immunoblotting analysis and in

sporozoite neutralization assays (Knight et al. 1996). Thus

the basis for a common vaccine component for T. annulata

and T. parva is available and this paper describes the results

of two reciprocal vaccination experiments using recombi-

nant SPAG-1 and p67.

MATERIALS AND METHODS

Antigen preparation

SPAG-1 production

Almost the entire SPAG-1 polypeptide, comprising residues

20±885 (full-length protein comprises 907 residues) was

expressed with a 6´ histidine tag at the N terminus (Hochuli

et al. 1989). Brie¯y, an EcoRV±BanII fragment of SPAG-1

cDNA (Hall et al. 1992) was end-®lled and cloned into the

SmaI site of the expression vector pQE32 (Qiagen GmbH,

Germany). Recombinant plasmid was transformed into

Escherichia coli M15(pREP4) and the insert DNA was

con®rmed to be in-frame and in the correct orientation by

double stranded sequencing across the junctions (data not

shown).

Fusion proteins were puri®ed on Ni-NTA resin, under

denaturing conditions, from lysates of M15(pREP4) con-

taining recombinant plasmids, prepared according to the

supplier's instructions (Qiagen). Approximately 3 ml frac-

tions were collected and analysed by SDS-PAGE and

Western blotting. Fractions containing puri®ed protein

were concentrated and desalted using an Ultrafree-15

column (Millipore Corp., Bedford, MA, USA). Protein

concentrations were quanti®ed using the BCA protein

assay kit (Pierce Chemical Co., Rockford, IL, USA).

p67 production

The p67 was produced as an N-terminal fusion with the NS1

protein of the in¯uenza A virus as described (Musoke et al.

1992).

Adjuvant

The mineral-oil containing adjuvant, RWL (kindly supplied

by P®zer Central Research, Sandwich, UK) was used in

these experiments. The antigen was mixed manually in

RWL at a ratio of 1 : 4 until a stable emulsion was obtained.

The antigen/RWL emulsion was used immediately after

preparation.

Immunization and challenge

The vaccination trial involving a T. annulata challenge

(experiment 1, Table 1) took place at the Centre for Tropical

Veterinary Medicine (CTVM) in Edinburgh, UK whilst that

using a T. parva challenge (experiment 2, Table 2) was

performed at The International Livestock Research Institute

(ILRI, Nairobi, Kenya). Both trials used three groups of

Friesian calves. In experiment 1, each group was composed

of six animals aged 3 months, whilst for experiment 2, group

A consisted of 10 and groups B and C seven animals aged

6±8 months. Each group received three subcutaneous inocu-

lations, given at monthly intervals comprising: groups 1 and

A, 450 mg His6-SPAG-1 in RWL; groups 2 and B, 450 mg

NS1-p67 in RWL; groups 3 and C, PBS-RWL control.

Fourteen days after the third immunization the calves

were challenged with either an estimated LD50 (0´2 tick

equivalents) of T. annulata Hisar sporozoite stabilate 52

(experiment 1) or an estimated LD70 of T. parva Muguga

sporozoite stabilate 4133 (experiment 2). Serum was col-

lected regularly pre- and postchallenge.

Clinical response to sporozoite challenge

Following challenge, daily examinations were performed to

look for signs of infections and rectal temperatures were

taken. From day 4 (experiment 1) and day 5 (experiment 2),

daily lymph node biopsies were taken until macroschizonts

were seen (to de®ne the prepatent period) and then three

times weekly thereafter and the parasitosis estimated. Blood

smears were taken regularly to estimate piroplasm parasi-

taemias. White blood cell counts and packed cell volume

(PCV) measurements were performed three times a week.

Animals suffering severe clinical disease were humanely

euthanased. The estimation of clinical severity in both

R.Hall et al. Parasite Immunology

q 2000 Blackwell Science Ltd, Parasite Immunology, 22, 223±230224

diseases relies upon the judgement of the respective veter-

inarians (Professor C.G.D.Brown for T. annulata and Dr S.

Morzaria for T. parva) but is largely based on the presence

of sustained fever combined with other symptoms most

notably inappetance and dyspnoea. In the case of T. annu-

lata, severity is most often accompanied by high (i.e > 50%)

piroplasm parasitaemia whilst a high degree (> 5%) of

schizont parasitosis is the best diagnostic correlate of the

severity of T. parva infections.

Reactions to T. parva challenge were graded as mild

(MR), moderate (MOR) or severe (SR) using an established

system described previously (Anonymous, 1989). Brie¯y,

MRs are essentially asymptomatic and are de®ned as having

a schizont parasitosis of less than 1% with mild fever for less

than four days followed by full recovery; MORs have mild

transient clinical signs (usually loss of appetite) with a

schizont parasitosis in the range 1±10% plus pyrexia lasting

no more than 9 days, followed by recovery; SRs have severe

clinical signs (inappetence, lethargy, rapid weight loss,

dyspnoea), requiring euthanasia with a schizont parasitosis

of greater than 10% and pyrexia for longer than 9

days usually accompanied by high (> 5%) piroplasm

parasitaemia, Although high piroplasm parasitaemia

(greater than 5%) are correlated with severe reactions in

T. parva, occasionally with very rapid dissemination of

schizonts animals may die before the high parasitaemia is

observed. Recovery was de®ned as disappearance of schi-

zonts from lymph nodes and a temperature below 39´58C. In

evaluation of immunity to challenge, MRs and MORs were

considered immune and SRs as susceptible. It was decided

that the spectrum of reactions observed in the T. annulata

infections did not lend itself to a similar system of

classi®cation.

Serological response

Experiment 1: T. annulata

Serum was sampled throughout the course of the experiment

and assayed for anti-SPAG-1 antibodies by ELISA, using a

method adapted from Hunt & Hall (1993). Serial dilutions of

all the sera collected were used to probe 96-well plates

coated with either GST-SPAG-1 or GST alone to act as a

negative control. HRP conjugated to antibovine IgG diluted

Volume 22, Number 5, May 2000 Cross-protection induced by sporozoite antigens SPAG-1

q 2000 Blackwell Science Ltd, Parasite Immunology, 22, 223±230 225

Table 1 Experiment 1: clinical reactions of calves immunized with SPAG-1 and p67 and challenged with T. annulata

Group Calf aPyrexia bMa cPiro Max Min wbc E R

% Piros ´ 103/ml

1 SPAG-RWL 2 H 8 9 12 15´2 2´6 ± 20

4 H 8 8 12 4´8 4´0 ± 17

6 H 8 6 12 35´5 2´8 ± 24

1 H 5 8 9 52´3 2´6 16 ±

3 H 7 6 12 71´1 3´0 19 ±

5 H 8 6 12 47´9 2´5 16 ±

Mean 7´3 7´2 11´5 37´65 2´92

2 p67-RWL 10 H 5 6 12 23´0 2´4 ± 27

11 H 5 7 9 40´0 3´0 ± 27

12 H 6 6 12 54´0 4´6 ± 24

7 H 7 5 12 48´0 1´8 23 ±

8 H 5 5 12 61´0 3´4 15 ±

9 H 7 5 12 66´0 1´5 18 ±

Mean 5´8 5´7 11´5 48´66 2´78

3 PBS-RWL 19 H 5 6 12 51´0 1´7 15 ±

20 H 5 6 12 70´1 2´2 15 ±

21 H 5 5 9 55´1 2´2 15 ±

22 H 5 5 12 30´5 3´8 23 ±

23 H 5 5 9 54´4 2´0 16 ±

24 H 5 5 9 70´6 2´6 14 ±

Mean 5´0 5´3 10´5 55´28 2´42

aPyrexia, ®rst day fever is greater than or equal to 39´58C (also termed the incubation period); bMa, day macroschizonts ®rst observed (also termed

the prepatent period); cPiro, day piroplasms ®rst observed; wbc, white blood cells; E, day euthanased; R, day recovered (de®ned as day when

temperature fell below 39. 58C).

1 : 1000 was used as the detection system and the substrate

comprised 0´25 M Na2HPO4, 50 mM citric acid, 0´05%

Tween 20, 10 mM orthophenylendiamine and 0´02% hydro-

gen peroxide (30% w/v). Western blot analysis was used to

perform epitope mapping studies. The sera were used, at a

dilution of 1 : 50, to probe Western blots of gels containing

1 mg of each of the pGEX constructs of SPAG-1, as

previously described (Boulter et al. 1994).

Experiment 2: T. parva

Serum antibodies speci®c for NS1/p67 were measured

by indirect ELISA following a method described (Katende

et al. 1998). Brie¯y, p635, a soluble form of NS1/p67 was

coated on to the microtitre plates at 8 ng/ml. The plates

were blocked with 5% casein before introduction of test sera

diluted at 1 : 200. The bound antibodies were revealed

by antibovine IgGs conjugated to HRP and ABTS as

chromogen.

Statistical analysis

The Mann±Whitney U-test was used for comparing

the incubation period, the prepatent period, days to ®rst

piroplasm and the minimum white blood cell counts. To

compare the maximum percentage piroplasm parasitaemia,

data were arcsine transformed and paired t-tests applied.

Signi®cance at the 5% level was used to de®ne differences

between the groups.



Table 2 Experiment 2: clinical reactions of calves immunized with SPAG-1 and p67 and challenged with T. parva

Group Calf aPyrexia bMa cPiro Max Min wbc E R dType of ECF reaction

% Piros ´ 103/ml

A SPAG-RWL BR 353 11 11 13 < 0´1 5´7 13 MR

BR 352 10 9 12 4´8 7´2 21 MR/MOR

BR 369 10 9 13 4´6 3´3 19 MOR

BR 354 11 7 13 1´0 1´4 22 MOR

BR 346 9 8 13 6´6 0´5 18 SR

BR 349 10 7 12 13´0 2´6 20 SR

BR 357 10 8 12 12´8 1´9 18 SR

BR 360 10 7 12 11´8 0´2 22 SR

BR 363 9 9 12 12´0 0´5 19 SR

BR 366 10 7 12 13´6 2´1 19 SR

Mean 10 8´2 12´4 8´03 2´54

B p67-RWL BR 347 17 12 14 0´2 12´6 18 MR

BR 361 10 8 13 < 0´1 5´3 17 MR

BR 367 13 8 14 < 0´1 6´8 18 MR

BR 350 10 8 14 1´0 4´2 16 MR

BR 355 9 9 13 8´6 0´3 21 SR

BR 358 9 9 14 11´6 0´4 21 SR

BR 364 10 7 13 16´0 0´6 17 SR

Mean 11´14 8´7 13´6 5´37 4´31

C PBS-RWL BR 351 8 7 13 2´2 1´5 18 MOR

BR 348 9 8 12 19´8 0´4 19 SR

BR 356 10 7 13 4´8 0´8 18 SR

BR 359 8 7 13 7´2 0´3 17 SR

BR 362 9 7 12 10´4 0´3 20 SR

BR 365 10 7 13 11´0 0´4 19 SR

BR 368 11 9 13 15´6 3´0 21 SR

Mean 9´29 7´4 12´7 10 0´96

aPyrexia, ®rst day fever is greater than or equal to 39´58C (also termed the incubation period); bMa, day macroschizonts ®rst observed (also termed

the prepatent period); cPiro, day piroplasms ®rst observed; wbc, white blood cells; E, day euthanased; R, day recovered (de®ned as day when

temperature fell below 39. 58C). dMR, mild reactor and recovered; MOR, moderate reactor and recovered; SR, severe reactor and euthanasia; MR/

MOR, severity between MR and MOR. MR, MR/MOR and MOR reactors are considered protected. The reactions were de®ned on a combination

of criteria that included the duration of pyrexia, parasitosis, parasitaemia, total white blood cell count and clinical conditions.

RESULTS

Levels of protection induced

Experiment 1: T. annulata challenge

All 18 animals developed classical tropical theileriosis

symptoms upon challenge with T. annulata, but of varying

intensities (Table 1). Notably, all six of the RWL adjuvant

control calves (group 3) had to be euthanased. In contrast,

three of six of the SPAG-1 and three of six of the p67

immunized calves recovered indicating that both antigens

offer a degree of protection. The SPAG-1 immunized group

appeared to be the best protected to challenge, as assessed

by several parameters. Thus the prepatent period (de®ned as

the day to ®rst schizont observed) is signi®cantly increased

to a mean of 7´2 days (median 7 days) compared to 5´3

(median 5) days for the RWL controls (P� 0´0202). The

prepatent period of the p67 group (mean 5´7, median

5´5 days) is not signi®cantly different to the RWL control

group. Similar trends are observed when analysing the mean

incubation period (day temperature ®rst exceeds 39´58C)

with values of 7´3, 5´8 and 5´0 days for the SPAG-1, p67 and

RWL controls, respectively. The difference between the

SPAG-1 and control groups is signi®cant (P< 0´025). Whilst

the RWL and P67 groups are not statistically different in

their incubation times the mean temperature of both the p67

and SPAG-1 groups stays 0´4±1´18C below the RWL group

from day 5±13 (Figure 1), which is statistically signi®cant

(P< 0´001, Student's t-test). Also the mean piroplasm para-

sitaemias (37´65%, SPAG-1; 48´66%, p67; 55´28%, control)

are concordant with this protective trend although these

values are not statistically different. However, the mean

parasitaemias of the three euthanased animals within the

SPAG-1 group are signi®cantly different (P< 0´05) from the

means of the three recovered animals within the group. Of

greater importance is the level of piroplasm parasitaemia on

days 12 and 14 (Figure 2). On both days, the SPAG-1 and

p67 values are signi®cantly lower than the corresponding

RWL treated animals. Furthermore, if the cumulative values

for piroplasm parasitaemia, schizont score, daily fever and

percentage PCV drop are calculated, the SPAG-1 group are

lowest for all four measurements (not shown). The p67

group is intermediate and the controls are highest (i.e. least

protected) by these criteria. Since the p67 group is consis-

tently better by several criteria than the RWL controls, we

conclude that this T. parva antigen gives some cross-

protection against T. annulata challenge.

Experiment 2: T. parva challenge

In this experiment, all 24 cattle became infected with

T. parva (Table 2). In the nonimmunized RWL adjuvant

control group (group C), six of seven cattle underwent

severe reactions (SR) and 1/7 had a moderate reaction

(MOR). Thus the challenge with a 1 : 80 dilution of

stabilate 4133 gave an LD86. In contrast, four of seven of



5 8 9 10 11 12 13 14

Days

41.5

41.3

41.1

40.9

40.7

40.5

40.3

40.1

39.9

39.7

39.5

Tem

per

atu

re (

° C)

Figure 1 Mean temperature in calves vaccinated with SPAG-1 (B),

p67 (O) and RWL control (´) and challenged with T. annulata

(experiment 1).

2.54.2

23.3

13.3

18.3

45

60

50

40

30

20

10

0SPAG P67 RWL SPAG P67 RWL

Days post-challenge

12 14

Perc

ent

Figure 2 Mean piroplasm parasitaemia days 12 and 14 in calves

vaccinated with SPAG-1, p67 and RWL control and challenged with

T. annulata (experiment 1).

the NS1/p67-immunized cattle in Group B had mild reac-

tions (MR) demonstrating protection in approximately 57%

of the animals. The parasitaemia levels of the p67 MR

calves are signi®cantly lower than the levels in the SR

animals of the same group (P� 0´0014). Analysis also

shows that the RWL control group has signi®cantly higher

parasitaemia levels than those of the p67 MR calves

(P� 0´012). In the SPAG-1 group of 10 calves, one had a

mild reaction, three had moderate reactions and six had

severe reactions. The mean parasitaemias of the SPAG-1

MR/MOR group are signi®cantly different from the SPAG-

1 SR (P� 0´0013) and the RWL (P� 0´046) groups. The

MR p67 group and MR/MOR SPAG-1 group white blood

cell counts, a more important indicator of disease severity in

ECF than tropical theileriosis, were also signi®cantly higher

(P< 0´05) than the corresponding SR group. The average

prepatent period is increased in the SPAG-1 (mean of

8´2 days) and p67 (mean of 8´7 days) immunized groups

relative to the PBS-RWL controls (mean 7´4 days). By

several criteria, the SPAG-1 group also shows evidence of

protection against T. parva challenge but to a lower degree

than the group immunized with NS1/p67. Thus, this experi-

ment provides evidence of some degree of a cross-protective

effect of SPAG-1 to T. parva challenge.

Serum responses of immunized animals

In experiment 1 (T. annulata challenge), signi®cant levels of

anti-SPAG-1 antibodies assayed by ELISA were generated

by day 56 in the SPAG-1 RWL immunized group and had

further risen by day 72. The anti-SPAG-1 pro®le was similar

in the p67 RWL immunized group although the titres were

lower (data not shown). In experiment 2 (T. parva chal-

lenge), all animals that survived had signi®cant antibody

titres from day 30 to p67 (data not shown).

DISCUSSION

Designing recombinant vaccines against parasites of all

types is proving to be a major challenge. Indeed, currently

no useable antiprotozoan subunit vaccine exists although

considerable progress is now being made in the malaria ®eld

and the prospects of eventual success are extremely positive.

Progress has been somewhat slower in bovine theileriosis

but advances have been made in both Theileria annulata and

Theileria parva where the prime focus has been the surface

antigens of the sporozoites. In one previous study of

Theileria annulata, a C terminal fragment (called SR1) of

SPAG-1 was linked to hepatitis B core antigen (HBcAg) as a

source of powerful T cell help. Multiple immunizations of

this construct in saponin resulted in high titres of sporozoite

neutralizing antibodies plus a signi®cant anti-SPAG-1 CD4

T cell response. Following sporozoite challenge, the vacci-

nated group had a delay in the prepatent period to macro-

schizont appearance, a reduced piroplasm parasitaemia and

4 out of 4 calves survived whilst 2 out of four control

animals (which received HBcAg and saponin) died (Boulter

et al. 1995). Our results reported here using a different

regimen con®rm that SPAG-1 is indeed protective against T.

annulata challenge with three out of six vaccinated animals

surviving, whereas all six controls died (Table 1). Other

indicators of a protective effect such as a delay in prepatent

period and an extension of the incubation period were also

observed (Table 1).

The Theileria parva p67 sporozoite antigen has been

expressed in a number of systems including E. coli, bacu-

lovirus, vaccinia and Salmonella dublin. (Musoke et al.

1992, Nene et al. 1996, Heussler et al. 1998, Honda et al.

1998). In the E. coli system the antigen has been used in

vaccination trials when expressed as a C-terminal fusion to

the nonstructural protein 1 (NS1) of in¯uenza virus and as a

his6 tag product. The ®rst published trial was using the NS1

fusion where nine indigenous Boran (Bos indicus) cattle

were immunized ®ve times with 3% saponin as the adjuvant

(Musoke et al. 1992). Six of the animals were protected

from an LD70 homologous challenge with T. parva Muguga.

Protection of four of the six was complete showing no

evidence of clinical reaction whilst the other two underwent

a mild reaction. The other three p67 immunized calves had

severe reactions as did all 10 controls. The mechanism of

protective immunity was unclear. All the nine animals

developed neutralizing antibodies but the titres did not

correlate with protection. The authors reported that two of

the nonreactors were PCR negative using T. parva speci®c

primers on lymph node biopsy material obtained 60 days

after the challenge suggesting that sterilizing immunity had

been induced. An important follow up investigation repro-

duced the initial results by observing protection in seven of

12 calves (two of which were completely protected) against

homologous challenge (Nene et al. 1996). More impor-

tantly, the authors reported that they protected six of 11

calves (one completely) against an heterologous challenge

with a stock (T. parva Marekibuni) which is not cross-

protective by infection and treatment. From the current data,

the protective effect of p67 against homologous T. parva

challenge is con®rmed with prevention of a severe ECF

reaction in four of seven calves (Table 2).

SPAG-1 and p67 antigens share signi®cant sequence

homology, cross-react serologically and antibodies against

one antigen will neutralize sporozoites of the other (Katzer

et al. 1994, Knight et al. 1996). Indeed, epitope mapping

studies performed using the sera generated in experiment 1 (of

this study) con®rmed that the N- and C-termini of SPAG-1



contain the dominant epitopes as reported (Knight et al.

1996). Particularly noteworthy was the fact that the antip67

sera recognize predominantly N- and C-terminal SPAG-1

constructs. This high degree of homology and serological

cross-reactivity led us to the possibility of cross-species

protection and the prospect of a common subunit vaccine

against tropical theileriosis and East Coast fever which

would be a highly desirable commodity. Therefore, we

performed the reciprocal immunization and challenge

experiments with SPAG-1 and p67 sporozoite antigens

from T. annulata and T. parva, respectively. The results

showed that p67 protected three out of six animals from T.

annulata challenge (the same as SPAG-1) and signi®cantly

reduced the mean temperature and parasitaemia over several

days (Table 1, Figures 1 and 2). A cumulative analysis of

piroplasm parasitaemia, PCV, schizont parasitosis and fever

indicated that the effect of p67 was not so protective as

SPAG-1 in the T. annulata challenged group. Conversely

SPAG-1 prevented a severe ECF reaction in four out of 10

calves (cf. six of seven controls reacted severely) (Table 2).

Thus, both antigens provide some cross-species protection.

The existence of cross-species protection within a single

host is perhaps unexpected as one would expect selection to

act against cross-protective epitopes. However, this is pos-

sibly explained by the fact that these two parasites rely on

tick vectors which have nonoverlapping geographical dis-

tributions (except in Sudan) and are thus effectively isolated

from one another. At a practical level, this type of shared

immunological identity suggest that common vaccine may

be feasible which could be economically bene®cial in terms

of production costs. In addition, global warming vector

distributions may change leading to a greater overlap of

the two diseases in the future, in which circumstances, a

common vaccine would be ideal.

ACKNOWLEDGEMENTS

Thanks are due to Andy Tait for constant support and

encouragement and the team at the Division of Protozool-

ogy, CTVM, University of Edinburgh for their enormous

enthusiasm. This work was funded by the European Union

(contract numbers IC18-CT95±0003 and IC18-CT 95 004)

and the EU ICTTD network (contract number IC18-CT95±

0009), the Department for International Development and

the BBSRC. This study constitutes ILRI publication number

990221.

REFERENCES

Anonymous (1989) Nomenclature in Theileria. In Theileriosis in

Eastern, Central and Southern Africa, Proceedings of a Workshop

on East Coast Fever Immunisation held in Lilongwe, Malawi,

20±23 September, 1988, ed. T.T. Dolan, pp. 187±188, ILRAD,

Nairobi

Boulter N.R., Brown C.G.D., Kirvar E. et al. (1998) Different vaccine

strategies used to protect against Theileria annulata. Annals of the

New York Academy of Sciences 849, 234±246

Boulter N.R., Glass E.J., Knight P.A. et al. (1995) Theileria annulata

sporozoite antigen fused to hepatitis B core antigen used in a

vaccination trial. Vaccine 13, 1152±1160

Boulter N. & Hall R. (1999) Immunity and vaccine development in the

bovine theilerioses. Advances in Parasitology 44, 42±97

Boulter N., Knight P.A., Hunt P.D. et al. (1994) Theileria annulata

sporozoite surface antigen (SPAG-1) contains neutralizing determi-

nants in the C terminus. Parasite Immunology 16, 97±104

Dolan T.T. (1989) Theileriasis: a comprehensive review. Revue

Scienti®que et Technique Of®ce International Des Epizooties 8,

11±36

Gentschev I., Glaser I., Goebel W et al. (1998) Delivery of the p67

sporozoite antigen of Theileria parva by using recombinant

Salmonella dublin: secretion of the product enhances speci®c

antibody responses in cattle. Infection and Immunity 66, 2060±

2064

Hall R., Hunt P.D., Carrington M et al. (1992) Mimicry of elastin

repetitive motifs by Theileria annulata sporozoite surface antigen.

Molecular and Biochemical Parasitology 53, 105±112

Heussler V.T., Taracha E.L.N., Musoke A., Duchateau L. & McKeever

D.J. (1998) Immunisation with live attenuated Salmonella dublin

expressing a sporozoite protein confers partial protection against

Theileria parva. Vaccine 16, 834±841

Hochuli E., Dobeli H. & Schacher A. (1989) New metal chelate

adsorbent is selective for proteins and peptides containing neigh-

bouring histidine residues. Journal of Chromatography 411, 177±

184

Honda Y., Waithaka M., Taracha E.L.N. et al. (1998) Delivery of

theileria parva p67 antigen to cattle using recombinant vaccinia

virus: IL-2 enhances protection. Vaccine 16, 1276±1282

Hunt P.D. & Hall R. (1993) Immunoblotting and enzyme linked

immunosorbent assay. In Protocols in Molecular Parasitology in

the Methods in Molecular Biology, ed. J.E. Hyde, pp. 397±406, The

Humana Press, Totawa, NJ

Irvin A.D. & Morrison W.I. (1987) Immunopathology, immunology

and immunoprophylaxis of Theileria infections. In Immune

Responses in Parasitic Infections: Immunology, Immunopathology

and Immunoprophylaxis, ed. E.J.L. Soulsb, pp. 223±273, CRC Press,

Boca Raton

Katende J., Morzaria S., Toye P et al. (1998) An enzyme-linked

immunosorbent assay for detection of Theileria parva antibodies in

cattle using a recombinant polymorphic immunodominant molecule.

Parasitology Research 84, 408±416

Katzer F., Carrington M., Knight P et al. (1994) Polymorphism of

SPAG-1, a candidate antigen for inclusion in a sub-unit vaccine

against Theileria annulata. Molecular and Biochemical Parasitology

67, 1±10

Knight P., Musoke A.J., Gachanja J.N. et al. (1996) Conservation of

neutralizing determinants between the sporozoite surface antigens of

Theileria annulata and Theileria parva. Experimental Parasitology

82, 229±241

Musoke A., Morzaria S., Nkonge C., Jones E. & Nene V. (1992) A

recombinant sporozoite surface antigen of Theileria parva induces

protection in cattle. Proceedings of the National Academy of Sciences

USA 89, 514±518

Musoke A., Nene V. & Morzaria S.P. (1993) A sporozoite-based

vaccine for Theileria parva. Parasitology Today 9, 385±388



Mutugi J.J., Ndungu S.G., Linyonyi A et al. (1991) Responses to a

vaccine trial for East Coast fever in two cattle herds at the Kenyan

Coast. Preventative Veteterinary Medicine 10, 173±183

Nene V., Musoke A., Gobright E. & Morzaria S. (1996) Conservation of

the sporozoite p67 vaccine antigen in cattle-derived Theileria parva

stocks with different cross-immunity pro®les. Infection and Immunity

64, 2056±2061

Radley D.E. (1981) Infection and treatment method of immunisation

against theileriosis. In Advances in the Control of Theileriosis, eds

A.D. Irvin, M.P. Cunningham, A.S. Young, pp. 227± 237, Martinus

Nijhoff, The Hague

Somerville R.P.T., Littlebury P., Pipano E et al. (1998) Phenotypic and

genotypic alterations underlying the attenuation of a Theileria

annulata vaccine cell line from Turkey. Vaccine 16, 569±575

Tait A. & Hall F.R. (1990) Theileria annulata: control measures,

diagnosis and the potential use of subunit vaccines. Revue Scien-

ti®que et Technique Of®ce International Des Epizooties 9, 387±

403

Williamson S., Tait A., Brown D et al. (1989) Theileria annulata

sporozoite surface antigen expressed in Escherichia coli elicits

neutralizing antibody. Proceedings of the National Academy of

Sciences of the USA 86, 4639±4643



reciprocal cross-protection induced by sporozoite antigens spag-1 from theileria annulata and p67...

Documents