Introduction

Tropical theileriosis is an infectious virulent inoculable, noncontagious disease of cattle (Robinson 1982). The causative agent is Theileria annulata, which is transmitted after cyclical development in the tick of the genus Hyalomma (Uilenberg 1981). The endemic area of T. annulata stretches out from the Mediterranean coastal regions in Southern Europe, Northern Africa, including Mauritania and surroundings of the Nile into Central Sudan and Middle East to South of the Caucasus splitting around Himalaya to India and China in Asia (Dolan 1989). Tropical theileriosis, also known as Mediterranean fever, causes losses in the productivity in indigenous as well as fatalities in imported foreign breeds and their crosses in endemic areas. The indirect fluorescence antibody test (IFAT) has successfully been used through decades to detect antibodies against Theileria infection in cattle (Burridge and Kimber 1973) and has been reported to be more sensitive than examination of blood smears in the field (Dhar and Gautam 1977; Darghouth et al. 1996). However, it is tedious and subjective, and the major drawback is the observed cross-reactions between Theileria species (Burridge et al. 1974; Kiltz et al. 1986). An enzyme-linked immunosorbent assay (ELISA) because of its accessibility to standardization and easy application would fulfill the requirements for epidemiological surveys when compared to the IFAT. To date, several ELISAs based on recombinant proteins have been developed for detection of infection with Theileria parva (Katende et al. 1998), T. annulata (Gubbels et al. 2000), T. lestoquardi (Bakheit et al. 2006a), and Theileria sp. (China) (Miranda et al. 2006a). The characterization of T. annulata surface protein (TaSP; Schnittger et al. 2002a) led to its application in indirect ELISA (Bakheit et al. 2004), which was validated using field sera and used in epidemiological surveys in Sudan (Salih et al. 2005a, b). This paper reviews the TaSP ELISA and the TaSP molecule.

Identification and molecular characterization of TaSP

The TaSP protein was identified by screening a T. annulata complementary deoxyribonucleic acid expression library with antischizont antiserum (Schnittger et al. 2002a). Subsequent sequencing identified it as the T. annulata homologue of T. parva PIM (Toye et al. 1995). TaSP occurs as a single-copy gene and is expressed in the sporozoite and schizont stages of the parasite. Homolgues of the gene were shown to exist also in other Theileria species namely, T. lestoquardi and Theileria sp. (China) (Schnittger et al. 2002b), and to be expressed on the messenger ribonucleic acid level (Bakheit et al. 2006b; Miranda et al. 2006b); however, stage-specific transcription has not been analyzed. Nonetheless, several findings indicate that the protein is expressed in the schizont stage of T. lestoquardi. An antiserum raised in rabbit against the recombinant TaSP recognized a band of similar in size in both T. annulata- and T. lestoquardi-infected cell lysates in Western blots and gave a similar pattern of staining in both parasites in IFAT (unpublished data). An alignment of the derived amino acid sequences from different Theileria species as listed in Table 1 and found in GenBank is shown in Fig. 1, and percent identities are summarized in Tables 1 and 2 (Combet et al. 2000; http://www.ebi.ac.uk/clustalw/). Clearly, there are conserved domains at the N and C termini of the gene, flanking a highly polymorphic central region of the sequence.

Table 1 Sequence identities of the TaSP protein and its homologues in different Theileria species calculated using Clustal W alignment (Combet et al. 2000)
Fig. 1
figure 1

Clustal-W alignment of Theileria annulata TaSP and homologues from T. sp. (China), T. lestoquardi, and T. parva. Sequences from different Theileria species as listed in Table 1. Shaded aa: recombinantly expressed TaSP sequence; * residues in the column are identical in all sequences in the alignment; : conserved substitutions have been observed; . semiconserved substitutions are observed

Table 2 Overall sequence identity and identities of the conserved N and C termini of TaSP and its homologues as listed in Table 1

Characteristics of TaSP

Using the TopPred “Topology prediction of membrane proteins” (Claros and von Heijne 1994; http://bioweb.pasteur.fr/seqanal/interfaces/toppred.html), the TaSP protein is predicted to be a membrane protein with three membrane domains, with a large segment containing the polymorphic region of the protein and facing the host cytoplasm, a smaller segment facing the schizont cytoplasm, the N terminus directed toward the parasite cytoplasm, and the C terminus toward the host cytoplasm (Fig. 2). The antiserum raised in rabbit against the recombinant TaSP protein was used for investigations on the subcelluar localization of the protein in schizont-infected cells, proving the localization to the parasite membrane. As determined by wide-field microscopy, detection of anti-TaSP antibody binding visualizes the schizont in the infected cells showing globular domains connected by filamentous structures located in close proximity to the host cell nucleus (Fig. 3a). Microinjection of fluorochrome-labeled anti-TaSP antibody into the cytoplasm of schizont-infected cells resulted in binding of the antibody to the outer surface of the parasite (Fig. 3b), and confocal imaging clearly underlined the detection of membrane structures by the antiserum directed against TaSP (Fig. 3c,d). Moreover, the microinjection experiment proved that the polymorphic region of the protein is facing the host cell cytoplasm, as the antibody was generated using the recombinantly expressed polymorphic region of TaSP. The exposure of the polymorphic region to the host cell cytoplasm also underlines the possibility of processing of this molecule by the host cell for major histocompatibility complex presentation, resulting in anti-TaSP antibody production by the infected host.

Fig. 2
figure 2

Schematic of the membrane topology of TaSP as predicted by TopPred (Claros and von Heijne 1994). N terminus with membrane domain 1: aa 1–21; membrane domain 2: aa 203–223; membrane domain 3: aa 261–281; segment between membrane domain 1 and 2, exposed to host cell cytoplasm and containing the polymorphic region (wavy line): aa 22–203; segment between membrane domain 2 and 3 and exposed to schizont cytoplasm: aa 224–260; C terminus exposed to host cell cytoplasm: aa 282–312

Fig. 3
figure 3

Subcellular localization of TaSP in Theileria annulata infected cells. a Wide-field microscopic detection of anti-TaSP antibody binding (green signal), showing the localization of the schizont in close vicinity and around the host cell nucleus (red signal). b Fluorescently labeled anti-TaSP antibody was microinjected into the cytoplasm of a T. annulata-infected cell, resulting in binding of the antibody to the parasite membrane (green signal). c Confocal image of a T. annulata-infected cell. TaSP (green signal), host cell, and parasite nuclei (red signal), d represents a close-up of the macroschizont. Size bars: a, b, c = 10 μm; d = 1 μm

The function of the TaSP protein in the parasite is unknown to date. Theileria schizonts are unique among protozoan parasites with their ability to transform their host cells. Within the leukocyte, the schizont undergoes synchronous division with the host cell. Transformation is independent of antigenic stimulation or growth factors, only depending on the parasite’s presence within the host cytoplasm. Importantly, elimination of the parasite leads to a resting phenotype and, subsequently, to apoptosis (Dobblaere and Heussler 1999; Heussler 2002). This reversibility of transformation indicates that the parasite’s influence on the host cell is not due to permanent changes in the host cell’s genome, leading to transformation. In contrast to other apicomplexan parasites like Plasmodium, Eimeria, and Toxoplasma, Theileria intracellular stages are lacking a parasitophorous vacuole and vesicular network (Shaw et al. 1991; Shaw and Tilney 1992). The fact that Theileria schizonts reside free in the cytoplasm offers several possibilities to interfere with host cell signaling pathways. Proteins on the outer membrane surface of the parasite or secreted proteins represent ideal candidates for parasite–host cell interactions. Because TaSP is a transmembrane protein with major portions exposed to the host cytoplasm, it represents a parasite protein that may interact with host cell proteins, and thus possibly be involved in upholding the transformed state of the host cell and thus maintenance of the transforming schizont stage to a certain period of time. This is supported by the absence of TaSP transcripts in the piroplasm (Schnittger et al. 2002a), a stage in the Theileria life cycle that comes after the transformation cycle ceases, and merozoites are released to infect erythrocytes.

Establishment of ELISA using a recombinant T. annulata surface protein (TaSP)

The polymorphic region of TaSP (gray-shaded sequence in Fig. 1) was recombinantly expressed as a His-tagged protein in Escherichia coli. Because this protein was recognized by serum from T. annulata-infected animals collected from different geographical areas, it was used to establish an indirect ELISA according to Office International des Epizooties guidelines (Bakheit et al. 2004). Like in the PIM ELISA (Katende et al. 1998), although highly variant sequences of the TaSP gene were found in a field study in Sudan (Mousa et al. 2005), the polymorphism in the sequence of TaSP seems to have no effect on the recognition of the protein by sera of animals exposed to different field strains. By testing 140 field sera in TaSP ELISA and a standard IFAT, the assay showed a sensitivity of 99.1% and specificity of 90.47% (Bakheit et al. 2004). No cross-reactions were found with Babesia species. Further validation of the assay was performed using sera from cattle exposed to tropical theileriosis in Sudan (Salih et al. 2005a). The reference-positive samples were from Theileria-infected populations and consisted of 80 cattle from an endemic area in Khartoum State, with high antibody titers in the IFAT. The reference-negative samples were taken from nonexposed populations and consisted of 120 cattle maintained under strict tick control at a commercial farm in Sudan. Detailed statistical analysis demonstrated that the TaSP ELISA is a useful test for the diagnosis of T. annulata infection in cattle under field conditions. A subsequent serological survey, in which 2,661 serum samples collected from different regions in Sudan were tested, showed an overall prevalence of tropical theileriosis at 33% but with a wide range of prevalence according to different regions in Sudan from 86.5% in Central Sudan to 17.9% in Western Sudan (Salih et al. 2005b; Table 3). These data correlated well with the distribution of the tick vector, as the highest values were observed in Central Sudan followed by Northern Sudan (Salih et al. 2004). It is interesting to note that a survey carried out on 162 samples from Central Sudan using polymerase chain reaction and reverse line blot also indicated a high prevalence of 65.4% (Ali et al. 2006), which was, however, less than the 86.5% obtained with TaSP ELISA. This may well reflect a situation in which low parasitemia in carrier animals may not be detectable in contrast to specific antibodies being measurable in the serum.

Table 3 Determination of prevalence rates of Theileria annulata infection in cattle from different locations in the Sudan during the year 2001–2002 using TaSP ELISA

Testing sequential sera collected from seven animals over the course of experimental infection of the ELISA showed that the TaSP ELISA detects infection with T. annulata well above the cutoff value 6 weeks postinfection and remain well detectable 12 weeks postinfection (Fig. 4).

Fig. 4
figure 4

Detection of T. annulata infection in experimentally infected animals using TaSP ELISA

It is interesting to note that antibody reactivity to the recombinant TaSP protein was also observed in animals infected with T. lestoquardi and Theileria sp. (China) (Miranda et al. 2004). The T. lestoquardi homologue (TlSP) was recombinantly expressed and shown to bind antibodies from serum of T. lestoquardi-infected animals (Bakheit et al. 2006b). Whether the respective homologues would be suitable for ELISA development, however, remains to be determined.

The TaSP protein has been shown to be highly immunogenic. A polyclonal antiserum was prepared by immunization of a rabbit with the recombinant TaSP and specific IgG antibodies were purified by affinity chromatography (HYDRA®-gelmatrix, Charles River Laboratories, Kisslegg, Germany). The purified antibody could be diluted 1:2 × 106 in ELISA for detection of the recombinant antigen and remained strongly reactive in immunoblots well beyond a dilution of 1:50,000. Moreover, other recombinant T. annulata proteins (TaD, Schneider et al. 2004, TaSE, Schneider et al. 2003, and rTamtHSP70, Schnittger et al. 2000) were tested in ELISA for antibody reactivity in serum from infected animals in comparison to recombinant TaSP. Results showed consistently higher antibody titers against the TaSP protein than against any of the others, whereby even no antibody reactivity was detected against TamtHSP70 (unpublished data). The presence of anti-TaSP antibodies in the serum of infected animals indicates that a protein expressed in the intracellular schizont stage of the parasite elicits a humoral immune response in the host. It is, however, unclear what the role of antibodies directed against this intracelluar stage may have in protective immunity.