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International Journal for Parasitology 40 (2010) 591598

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International Journal for Parasitology


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Identication of Theileria uilenbergi immunodominant protein for development of an indirect ELISA for diagnosis of ovine theileriosis q
Zhijie Liu a,b, Zijian Wang b, Hong Yin b, Jianxun Luo b, Bao Zhang b, Birgit Kullmann a, Jassim Abdo a, Dialeldin Salih a, Jabbar Ahmed a, Ulrike Seitzer a,*
Division of Veterinary Infection Biology and Immunology, Research Center Borstel, Parkallee 22, D-23845 Borstel, Germany Key Laboratory of Veterinary Parasitology of Gansu Province, Key Laboratory of Grazing Animal Diseases MOA, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China
b a

a r t i c l e

i n f o

a b s t r a c t
Theileriosis of small ruminants in the northwest of China is a protozoan disease that restricts the development of the livestock industry. The disease is caused by infection with Theileria uilenbergi and Theileria luwenshuni, both of which are transmitted by ixodid Heamaphysalis ticks. The development of serological tools as a means of integrated control of the disease is an urgent and important requirement. Here we describe the identication and partial recombinant expression of a T. uilenbergi immunodominant protein (TuIP), which was identied by immunoscreening of a merozoite cDNA library. Using the recombinant TuIP (rTuIP), a novel indirect ELISA was established using 329 negative serum samples to determine the cut-off value. The internal quality control revealed satisfactory stability and repeatability of the assay. Preliminary validation using 128 positive and 48 negative reference samples demonstrated that the rTuIP ELISA is able to detect T. uilenbergi infection with high sensitivity and specicity. No crossreactivity was found in sera from animals infected with Theileria lestoquardi, Babesia sp. China or Anaplasma ovis. Furthermore, circulating antibodies were detected in sera collected from endemic regions in China. Analyses of the antibody responses of experimentally infected animals demonstrated that tick infestation resulted in a sharply rising and stronger production of specic antibodies against TuIP while inoculation with infected blood induced an earlier production of TuIP-specic antibodies. The persistence of the TuIP-specic antibodies lasted more than 100 days p.i. These data indicate the usefulness of the TuIP antigen for the development of diagnostic methods and as a potential candidate for vaccine design. 2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Article history: Received 11 September 2009 Received in revised form 19 October 2009 Accepted 20 October 2009

Keywords: ELISA Immunodominant protein Theileria uilenbergi Theileriosis

1. Introduction Theileriosis of small ruminants caused by Theileria uilenbergi and Theileria luwenshuni (Yin et al., 2007) is a severe and often lethal disease, constituting a severe restriction for the development of the small ruminant livestock industry in the northwest of China, especially regarding the use of exotic animals (Luo and Yin, 1997). Ixodid Haemaphysalis ticks, Haemaphysalis qinghaiensis and Haemaphysalis longicornis have been demonstrated to be responsible for transmission of the disease (Yin et al., 2002; Li et al., 2007, 2009). Although a recent report of a protozoan isolated in Spain had molecular similarity at the 18S rRNA gene level to Chinese ovine Theileria (Nagore et al., 2004), the only disease reports to date are from China. Molecular phylogenetic studies have shown that the Chinese Theileria is closely related to the Theileria buffeli and
q Note: Nucleotide sequence data reported in this paper is available in the GenBank database under the Accession No. FJ467922. * Corresponding author. Tel.: +49 4573 188 413; fax: +49 4537 188 627. E-mail address: useitzer@fz-borstel.de (U. Seitzer).

Theileria sergenti groups (Schnittger et al., 2003; Yin et al., 2004). It is assumed that these closely related parasites share the common feature of exhibiting a less marked leucocytic phase and not being able to transform their host cells (Ahmed et al., 2006). Regarding the diagnosis of the disease, traditionally T. luwenshuni and T. uilenbergi infections are examined by observation of merozoites in Giemsa-stained blood smears under the microscope and/or observation of the clinical signs of the infected animals (Luo and Yin, 1997). However, these methods do not detect carrier status, are time consuming, not very sensitive and require operators with high expertise. To improve diagnostic methods, PCR (Yin et al., 2008), reverse line blot (RLB) (Schnittger et al., 2004) and loop mediated isothermal amplication (LAMP) (Liu et al., 2008b) have been developed. Although highly sensitive and specic, RLB and PCR are laborious and expensive, whereas LAMP as a novel developed method still needs to be validated. In contrast, serological diagnostic tools are not only relatively inexpensive, practical, easy to use and suitable for large scale surveys, but are also an important method for monitoring the antibody responses of infected animals. Thus, two indirect ELISA methods have been

0020-7519/$36.00 2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2009.10.011

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developed for diagnosis of theileriosis in China, one based on crude merozoite antigen (Gao et al., 2002), another based on the partially expressed Theileria lestoquardi recombinant heat shock protein 70 (rTIHSP 70) (Miranda et al., 2006a). There is still, however, a need for improvement, as the crude antigen ELISA is difcult to standardise, requires infection of animals for antigen preparation and is potentially cross-reactive with other related pathogens. On the other hand, the rTIHSP 70 gene used for the recombinant protein ELISA originated from T. lestoquardi and bears the risk of crossreactivity with other piroplasms infecting small ruminants. In the present study, a T. uilenbergi merozoite cDNA library (Liu et al., 2008a) was immunoscreened with pooled T. uilenbergi-positive sera with the aim of identifying antigenic and specic genes of T. uilenbergi. An antigenic gene termed TuIP was obtained and a partially expressed recombinant TuIP (rTuIP) was characterised and applied for establishment of a novel ELISA. The potential applicability of the ELISA was investigated by analysing serum from experimentally infected sheep and sera collected from endemic regions. 2. Materials and methods 2.1. Source of serum samples Experimental infections were conducted in the experimental animal facility of the Lanzhou Veterinary Research Institute in China and experimental sheep were purchased from a Theileria- and other protozoa-free area. Blood lms made from a drop of blood from the ear vein were Giemsa-stained and microscopically examined prior to experimental infection. Only sheep negative for haemoparasites were used for producing negative serum samples (sample No. 2201 and a further 329 negative samples collected during the years 20032008). Additional negative sera were collected from a slaughterhouse in Germany. Animal experiments with sheep in China and rabbits in Germany were performed only after formal ethics approval. Care and maintenance of animals was in accordance with institutional and governmental guidelines of the country (China or Germany) in which the experiments were carried out. Positive serum samples were obtained in a number of ways: (i) inoculation of sheep using blood-infected with T. uilenbergi Longde isolate (Yin et al., 2004; sera samples 1201, 1203, 1205) and Lintan isolate (Seitzer et al., 2008; sera samples 2203, 1236 and 1219); (ii) attachment of ticks collected in an endemic region (Lintan) (1410, 1411, 1412, 1250, 1240 and 1229). Positive sera of Babesia sp. and Anaplasma ovis were generated from Babesia sp. and A. ovis-infected sheep, whereas positive sera of T. lestoquardi used in this study were from an endemic region in Sudan (Bakheit et al., 2006). The reference positive samples were 128 eld serum samples collected from Lintan, China with a high O.D. value when tested by the merozoite antigen ELISA (Guo et al., 2007). Field samples from different endemic regions in the Gannan Tibet Region of Gansu Province were collected in June in 2004 and 2005 (Azitan of Zhouni County, n = 237; Ganjia of Xihe County, n = 194; Yangyong of Lintan County, n = 81). 2.2. Identication of the TuIP gene The construction of a cDNA library of T. uilenbergi from the merozoite stage has been described elsewhere (Liu et al., 2008a). The pooled serum samples from T. uilenbergi-infected sheep were used to conduct immunoscreening of the library. In order to remove Escherichia coli-reactive antibodies, the sera were rst adsorbed against E. coli XL1-Blue extracts bound to nitrocellulose membrane

(Schleicher and Schuell, Dassel, Germany). Plaques were generated by plating the library onto Luria broth (LB) plates according to the manual of the picoBlue immunoscreening kit (Stratagene, La Jolla, CA, USA). Phages were induced to express protein by placing nitrocellulose membranes soaked in 10 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) and dried on the surface of the plaques. Subsequently, membranes were washed in PBS and the non-specic binding sites blocked in PBS, 2.5% sh gelatin (Sigma, Deisenhofen, Germany). Serum was diluted 1:500 in PBS with 0.1% Tween 20 (PBST) containing 1% sh gelatin. Thereafter, lters were incubated with serum at room temperature for 2 h while shaking. After washing in PBST, a conjugate (donkey anti-sheep alkaline phosphatase-conjugated Fab fragments; Dianova, Hamburg, Germany) diluted 1:20,000 in PBST, 1% sh gelatin was incubated and lters then washed again as described above. Colorimetric development was conducted via incubation of the membranes in a substrate buffer that contained (Nitro-Blue Tetrazolium Chloride/5-Bromo-4-Chloro-30 -Indolyphosphate p-Toluidine salt (NBT/ BCIP) (Roth, Karlsruhe, Germany). A positively reacting plaque was isolated after repeated sub-plating and subjected to sequencing (Eurons MWG Operon, Ebersberg, Germany) using vector-specic primers (T3/T7, Stratagene, La Jolla, CA, USA). 2.3. Preparation of rTuIP The N-terminus of the identied gene sequence of 2,181 bp contained a unique BamHI restriction site from the kZAP vector sequence and a unique KpnI restriction site at nucleotides (nt) 1,0671,072; these restriction sites were used to sub-clone the N-terminal part of the gene into the pQE31 expression vector (Qiagen, Hilden, Germany), resulting in a recombinantly expressed protein of 382 amino acids with a predicted mol. wt of 417 kDa. This product was termed rTuIP. Additionally, the C-terminal sequence segment from nt 1,110 to 2,011 was sub-cloned into the pQE30 expression vector using BamH1 and SalI sites, which were introduced into the sequence by PCR amplication. This 326 amino acid protein had a predicted size of 37.3 kDa. Selected clones were checked to contain the insert by PCR and sequenced using pQE vector-specic primers (Qiagen, Hilden, Germany) (type III/IV forward 50 -CGGATAACAATTTCACACAG-30 and pQE reverse 50 -GTTCTGAGG TCATTACTGG-30 ). Histidine tagged recombinant protein was expressed in M15 E. coli and puried using NiNTA agarose beads under native conditions following the QiaExpressionist protocols (Qiagen, Hilden, Germany). This rst round of puried recombinant protein was applied to re-purication using the kta prime high system (Amersham Bioscience, Uppsala, Sweden) using 5 ml HiTrap columns packed with Ni Sepharose 6 Fast Flow. The Nichromatography was carried out according to the manufacturers instructions using the following chromatography programme: (i) washing of the column with 510 column volume (cv) H2O with a ow rate of 5 ml/min; (ii) equilibration with 10 cv application buffer (20 mM TrisHCl, pH 8.0, 8 M urea, 0.5 M NaCl, 5 mM imidazole); (iii) application of the sample to the column with a owrate of 1 ml/min; (iv) washing with 20 cv washing buffer (20 mM Tris HCl, pH 8.0, 8 M urea, 0.5 M NaCl, 5 mM imidazole) at a ow rate if 3 ml/min until the absorbance at A280 is constant; (v) collection of 1 ml fractions after application of 1 cv elution buffer (20 mM Tris HCl, pH 8.0, 8 M urea, 0.5 M NaCl, 1 M imidazole) with a linear gradient of 500 mM to 1 M imidazole until the protein peak appears at a ow rate of 1 ml/min. Collected fractions were analysed for the presence of protein using Western blots. Fractions containing the protein were pooled, dialysed against PBS and adjusted to a concentration of 0.2 mg/ml. This re-puried protein was used in all experiments with Western blot and ELISA. Recombinant protein expression was veried in Western blots using an anti-histidine antibody and T. uilenbergi-positive serum samples. Purity was as-

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sessed by Coomassie gel staining, and protein concentration was determined using the BioRadMicro-DC Assay kit (BioRad, Munich, Germany). 2.4. Preparation of rabbit anti-rTuIP anti-serum The puried TuIP recombinant protein was used to immunize three rabbits for generation of rabbit anti-rTuIP anti-serum (Charles River, GmbH, Kisslegg, Germany). Sera of three pre-immunized rabbits were tested by Western blots of the rTuIP protein, and the one showing minimal background reaction was chosen for immunization. The immunization regime covered four boosts each of 200 lg puried rTuIP protein at 2 week intervals, whereby the rst inoculation was conducted with FCA and the following three boosts with incomplete Freunds adjuvant. The successful production of the anti-TuIP anti-serum was veried by dot blot detection prior to animal bleeding. The rabbit was bled 10 days after the nal boost and the serum stored at 20 C until used. 2.5. Western blot analysis The preparation of merozoite crude antigen was carried out according to the protocol described by Gao et al. (2002). Optimal amounts of merozoite crude antigen and recombinant proteins were subjected to SDSPAGE using 12% polyacrylamide gels under reducing conditions and transferred to nitrocellulose membranes. To verify the expression and purication of the recombinant proteins, the RGS-His mouse anti-histidine antibody (1:4,000, Qiagen, Hilden, Germany) and secondary alkaline phosphatase (AP) conjugated goat anti-mouse IgG + IgM (H+L) antibody (1:10,000, Dianova, Hamburg, Germany) were used to detect the His-tag on the recombinant proteins. Merozoite crude antigen and rTuIP were Western blotted and probed with pre-immunization rabbit serum, rabbit anti-rTuIP anti-serum and rabbit anti-rTuIP anti-serum pre-absorbed with rTuIP (1:2,000 and 1:4,000) for detection of the protein. The secondary antibody for these experiments was AP-conjugated goat antirabbit immunoglobulin antibody (1:2,500; Dako, Glostrup, Denmark). All of the serum samples and the secondary antibodies were diluted in dilution buffer (1% BSA, 0.1% Tween 20 in PBS, 137 mM NaCl, 2.67 mM KCl, 3.2 mM Na2HPO4, 1.47 mM KH2PO4, pH 7.2). Binding of secondary antibody was detected with a BCIP/NBT substrate. 2.6. Establishment of rTuIP indirect ELISA protocol Serum 1229P, collected after observation of the piroplasms in Giemsa-stained blood smear at day 58 post inoculation in an experimentally infected sheep, served as the positive control. Negative control serum sample 1229N was collected from the same animal prior to infection. The optimum dilutions of the rTuIP antigen, positive serum 1229P, negative serum 1229N and the conjugate (peroxidase-conjugated Afnipure rabbit anti-sheep IgG (H+L, Dianova, Hamburg, Germany)) were checkerboard-titrated on a 96-well ELISA plate. The optimum dilution considered was the highest dilution of antigen/conjugate that still saturated the plate and gave maximum contrast in terms of O.D. between positive and negative sera. Thereafter, a standard indirect ELISA protocol was established. Coating was done by adding 100 ll antigen (TuIP) solution (0.5 lg/ml in carbonate bicarbonate buffer (pH 9.6) (Sigma, Deisenhofen, Germany)) into each well of 96-well ELISA plates (Maxisorp, Nunc, Glostrup, Denmark) except for four blank control wells lled with carbonate bicarbonate buffer only. The plates were incubated at 4 C overnight, then washed with PBS containing 0.05% (v/v) Tween 20 (pH 7.4) in three dispensing/aspiration cycles using a Bio-Tek 405 automated ELISA plate washer (Bio-Tek, Bad

Friedrichshall, Germany). Non-specic binding sites in each well were blocked using 200 ll 1% (w/v) BSA (Sigma, Deisenhofen, Germany) in PBS (pH 7.4) for 1 h under orbital shaking. The plates were washed again before 100 ll of sera diluted 1:400 in diluent buffer (1% BSA in PBS (pH 7.4)) were applied in duplicate for testing. Control positive (C+) and control negative (C) sera were applied in four replicates and four wells were always used as a conjugate control (CC), receiving only the diluent buffer. Plates were incubated for 1 h at room temperature on an orbital shaker, followed by a washing step. Then 100 ll of the conjugate horse radish peroxidase-labelled rabbit anti-sheep antibody (Dianova, Hamburg, Germany) diluted 1:15,000 in the diluent buffer were added to all wells and incubated. After washing, 100 ll of freshly prepared substrate solution [H2O2/tetramethylbenzi-dine (TMB, Sigma, Deisenhofen, Germany) in citric acid buffer (pH 4.0)] were dispensed into each well. Colour development was in the dark for 10 min and the reaction stopped by adding 100 ll 1 N H2SO4. The absorbance at 450 nm was measured using an ELISA reader (Expert 96, Asys Hightech, Overath, Austria; and Stat Fax 2100, Awareness Technology, Palm City, FL, USA). The obtained O.D. values were expressed as percent positivity (PP) of the internal positive control as follows: [(average O.D. value of testing samples average O.D. value of blank controls)/(average O.D. value of the positive controls average O.D. value of blank controls)] 100. The ELISA cut-off value, which served as the threshold between positive and negative sera, was determined as the mean PP value obtained from the 329 negative serum samples plus 2 S.D. 2.7. Application of the rTuIP indirect ELISA A total of 512 eld samples from three locations as described above was examined using the established ELISA. Based on the calculated specicity (Sp = #samples negative in both tests/total number of negative samples from both tests 100) and sensitivity (Se = #samples positive in both tests/total number of positive samples from both tests 100) of the rTuIP ELISA, the apparent prevalence (AP) and true prevalence (TP) were calculated, respectively, using the following formula:

AP number of positive samples=number of total samples TP AP Sp 1=Se Sp 1

3. Results 3.1. Identication of TuIP Sequence analysis of the clone isolated by immunoscreening of the merozoite library revealed an incomplete open reading frame (ORF) lacking a start codon with a size of 2,181 bp. This incomplete ORF encoded for a protein of 670 amino acids with a predicted mol. wt of 74.8 kDa. In addition, the clone contained 167 bp of the 30 untranslated region with 37 bp of a poly A tail. BLASTn searches within available Apicomplexa genome databases (http://www. ncbi.nlm.nih.gov/sutils/genom_tree.cgi?organism=euk) found no signicant similarity, while BLASTp searches showed the highest identity (Identities = 218/678 (32%), Positives = 331/678 (48%), Gaps = 55/678 (8%) to a hypothetical protein of Theileria parva (Gene ID: 3503410 TP01_0987) and its Theileria annulata orthologue (Identities = 216/681 (31%), Positives = 327/681 (48%), Gaps = 80/681 (11%), polymorphic antigen precursor-like protein, Gene ID: 3864034 TA16685) (Supplementary online material in Pain et al., 2005). Using a prediction server for antigenicity (http://immunax.dfci.harvard.edu/Tools/antigenic.pl) the average antigenic propensity for this protein was given as 1.0253, consisting of 27 antigenic determinants (Kolaskar and Tongaonkar, 1990).

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Fig. 1. Specicity and testing of cross-reactivity of the recombinant Theileria uilenbergi immunodominant protein (TuIP) using Western blot. Lane M, mol. wt marker; lanes 16, T. uilenbergi-positive serum samples Nos. 1201, 1203, 1205, 1410, 1411, 1412; lane 7, T. lestoquardi-positive serum Tl17; lane 8, Babesia sp. China-positive serum; lane 9, Anaplasma ovis-positive serum; lanes 10 and 11, negative serum; lane 12, donkey anti-sheep secondary antibody; lane 13, mouse anti-RGS-Hisantibody; lane 14, goat anti-mouse secondary antibody control. A specic band at approximately 50 kD was detected by the T. uilenbergi-positive serum samples and the anti-His-antibody, together with specic smaller bands most likely corresponding to breakdown products of the protein. No protein bands were detected with serum from animals infected with other small ruminant tick-borne pathogens.

Fig. 2. Specicity and testing of cross-reactivity of the recombinant Theileria uilenbergi immunodominant protein (TuIP) using ELISA. Sera 16, T. uilenbergi-positive serum samples Nos. 1201, 1203, 1205, 1410, 1411, 1412; sera 7, T. lestoquardipositive serum Tl17; sera 8, Babesia sp. China-positive serum; sera 9, Anaplama ovispositive serum; sera 10 and 11, negative serum. Each sample was diluted 1:200, 1:400 and 1:800. The cut-off of the ELISA is indicated by a horizontal line. As in the Western blot (Fig. 1), only sera from Theileria (China)-infected animals show binding of specic antibodies resulting at a signal above the established threshold.

Analysis of major histocompatibility complex I (MHC-I) binding epitopes based on bovine class I A20 specicity (http://www-bimas.cit.nih.gov/molbio/hla_bind) predicted four nonamer peptides with a dissociation half-time of 1,000 min (Parker et al., 1994). Thus, the identied gene encoded for a potentially antigenic protein of T. uilenbergi. The recombinantly expressed N- and C-terminal fragments of TuIP were tested for reactivity with positive sera, indicating that there was no apparent binding of antibodies to the 37.3 kDa C-terminal fragment. In contrast, the N-terminal partial recombinant protein, rTuIP with a calculated mol. wt of 41.7 kDa, could be recognised by T. uilenbergi-positive serum samples while no cross-reactivity was seen with serum samples of T. lestoquardi-, Babesia sp. China- and A. ovis-infected animals or negative sera using both Western blots (Fig. 1) and ELISA (Fig. 2). This indicated that the TuIP gene encodes a potential immunodominant protein of T. uilenbergi and it was thus termed T. uilenbergi Immunodominant Protein (TuIP) (Genbank Accession No. FJ467922). Immunization of a rabbit with rTuIP resulted in a specic antirTuIP anti-serum with a titer of 1:51,200 (data not shown). This anti-serum was used to identify the native TuIP protein in merozoite crude antigen of T. uilenbergi in Western blots. The results showed that the rabbit anti-TuIP anti-serum could specically recognise a single parasite protein band around 72 kDa in size (Fig. 3). Pre-absorption of the rabbit anti-rTuIP anti-serum with 3.5 lg of rTuIP protein inhibited the detection of this protein band, indicating the specicity of the reaction. Moreover, the pre-immunization rabbit serum was also negative. These data further conrmed the parasite origin and the immunodominant feature of the TuIP protein. 3.2. Establishment of the rTuIP indirect ELISA The optimal dilutions of serum samples and conjugate were checkerboard-titrated using known positive and negative serum samples diluted twofold from 1:100 to 1:3,200, while conjugate was diluted at 1:10,000, 1:12,500, 1:15,000 and 1:20,000. Since the serum sample dilution at 1:400 and conjugate dilution at 1:15,000 gave the maximum contrast in the O.D. value between

Fig. 3. The lysate of puried Theileria uilenbergi merozoites was blotted and tested for reactivity with rabbit anti-recombinant T. uilenbergi immunodominant protein (rTuIP) antibody. Lane M, mol. wt marker; lanes 1 and 2, 1:2,000 and 1:4,000 diluted rabbit anti-rTuIP anti-serum; lanes 3 and 4, 1:2,000 and 1:4,000 diluted rabbit antiTuIP anti-serum after blocking by 3.5 lg rTuIP; lanes 5 and 6, 1:2,000 and 1:4,000 diluted pre-immunized rabbit serum; lane 7, goat anti-rabbit secondary antibody control.

positive and negative samples, these conditions were used in all subsequent experiments (data not shown). The internal quality control was assessed by testing positive sample 1229P and negative sample 1229N in six different plates on two different days (Table 1). Similar values for the samples were
Table 1 Internal quality control results from the recombinant Theileria uilenbergi immunodominant protein (rTuIP) indirect ELISA by measuring six different plates on two different days. Plate number Day 1 1 2 3 1 2 3 C++ PP 108.05 3.41 94.17 2.04 104.25 3.91 97.19 1.34 104.31 2.61 107.43 5.60 C PP 3.88 0.25 3.10 0.24 4.34 0.28 3.15 0.43 4.27 0.49 4.24 0.33 CC PP -0.19 0.07 -0.24 0.16 -0.15 0.22 -0.08 0.18 -0.16 0.20 -0.04 0.10

Day 2

C++ PP, mean percent positivity (PP) value of the positive control; C PP, mean PP value of negative control; CC PP, mean PP value of conjugate control. Average values of four tests are given with S.D.

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Fig. 4. Preliminary validation of the recombinant Theileria uilenbergi immunodominant protein (rTuIP) ELISA using positive (s) and negative (d) reference serum samples. Only two negative samples were on or slightly above the established threshold, likewise only one of the positive samples lay below the cut-off value.

obtained throughout for PP values of C+, CC and C. The average PP value of the C+ was 102.57 3.15 with the maximum of 108.05 PP and the minimum of 94.17 PP, whereas the C was an average of 3.83 0.34 PP with a maximum of 4.34 PP and a minimum of 3.10 PP. The cut-off of the rTuIP ELISA was dened as the mean PP value of 329 negative serum samples plus two times the S.D. Before sampling, blood smears of all animals were examined under the microscope, conrming the absence of haemoparasites. Moreover, subsequent observation of these animals gave no indication that they were infected by protozoa before transportation to the laboratory. The average PP value obtained from these samples was 15.81, while the calculated S.D. was 8.55 PP, resulting in a cut-off of 32.9 PP. A preliminary validation of the rTuIP indirect ELISA was carried out by testing 128 positive reference samples according to previous investigations on sera collected from the eld using crude antigen ELISA (Guo et al., 2007) and 48 negative reference samples from uninfected sheep from Sudan (n = 12), Germany (n = 10) and China (n = 26). The results showed that among the 128 positive samples, 126 were tested positive, whereas 47 of 48 negative reference samples were negative, resulting in a calculated sensitivity and specicity of 98.44% and 97.92%, respectively (Fig. 4 and Table 2).

Fig. 5. Antibody response of six animals experimentally infected with Theileria luwenshuni against the recombinant Theileria uilenbergi immunodominant protein (rTuIP) as assessed by the rTuIP ELISA at three time-points (0, 27 and 47 days (d) p.i.). Using the established threshold, two animals (08516, 08455) showed some reactivity, two (08035, 08218) showed a low reactivity and two (08222, 08498) no reactivity to the rTuIP protein.

3.3. Analysis of the antibody response in experimentally infected animals using rTuIP ELISA Theileria luwenshuni is a closely related pathogen to T. uilenbergi. Both often occur together in theileriosis endemic regions in China (Yin et al., 2004). Therefore, the antibody response of T. luwenshuni-infected animals against rTuIP protein was separately investigated using the rTuIP ELISA. Six experimental sheep were

infected by blood inoculation with the Weiyuan strain veried by PCR to be T. luwenshuni (Yin et al., 2008). Serum samples were collected at days 0, 27 and 47 post inoculation. The successful infection of all the animals was proved by the observation of parasites in blood smears. The ELISA results showed that four animals (Nos. 08035, 08218, 08516 and 08455) produced antibodies against rTuIP (Fig. 5), although the O.D. values were conspicuously low compared with the results obtained with serum from T. uilenbergi-infected animals described below. Two of the animals (08222, 08498) were negative for antibodies against rTuIP applying the established threshold. To monitor the antibody response of animals experimentally infected with T. uilenbergi against rTuIP, the serum samples from different time-points during infection from six individual sheep were investigated. Among these animals, three (Nos. 2203, 1236 and 1219) were infected by inoculation of blood-infected with the Lintan strain, and another three (Nos. 1250, 1240 and 1229) were infected by feeding 200 H. qinghaiensis ticks from an endemic region (Seitzer et al., 2008). Infection of the animals was veried according to the observation of parasites in blood smears. The results of the ELISA demonstrated that the infection with infected blood induced antibodies binding to rTuIP in less than 14 days and increased from there, while tick infestation required in 2/3 cases more than 14 days to produce antibodies reacting with rTuIP. Although the titer of the antibodies was higher in tick-infected animals, in both infection models the rTuIP ELISA was able to detect specic antibodies more than 3 months p.i. This indicates that the test is a suitable tool for detection of early infection and monitoring of persistent infections with T. uilenbergi (Fig. 6). 3.4. Detection of circulating anti-rTuIP antibodies in eld samples from theileriosis endemic regions The eld serum samples randomly collected at three different locations (Azitan, Ganjia and Yangyong) in Gannan Tibet Region were tested by rTuIP ELISA. As shown in Table 3 and 229 out of 237 samples tested positive in Azitan, 175 out of 194 were positive in Ganjia and 74 out of 81 were positive in Yangyong. The respective apparent and true prevalences of theileriosis in these regions were calculated to be 96.62%, 90.21%, 91.36% and 98.12%, 91.47%, 92.67%, respectively (Table 3). 4. Discussion Previous phylogenetic studies on T. uilenbergi and T. luwenshuni revealed that they are most closely related to the non-transform-

Table 2 Preliminary validation of the sensitivity and specicity of the recombinant Theileria uilenbergi immunodominant protein (rTuIP) ELISA using positive and negative reference serum samples. Crude antigen ELISA Positive rTuIP ELISA Positive Negative Total 126 2 128 Negative 1 47 48 Total 127 49 176

Sensitivity of the rTuIP ELISA = 98.43% (number of samples positive in both tests/ total number of positive samples from both tests 100 = 126/128 100). Specicity of the rTuIP ELISA = 97.92% (number of samples negative in both tests/ total number of negative samples from both tests 100 = 47/48 100).

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Fig. 6. Antibody response of six animals experimentally infected with Theileria uilenbergi against the T. uilenbergi immunodominant protein (TuIP) over time as assessed by the recombinant TuIP (rTuIP) ELISA p.i. Sheep Nos. 1250, 1240 and 1229 were infected by feeding 200 adult Heamaphysalis qinghaiensis ticks collected from Lintan, China, Sheep Nos. 2203, 1236 and 1219 were infected by blood inoculation with T. uilenbergi Lintan isolate. There is a signicant difference in the antibody response between the two groups (one-way ANOVA, P < 0.05 for days 14, 58, 70 and 82 p.i.). PP, percent positivity; d, days.

Table 3 Detection of specic antibodies in 512 eld serum samples from China, using the recombinant Theileria uilenbergi immunodominant protein (rTuIP) ELISA. Locations Azitan Ganjia Yangyong
a

Positive 229 175 74

Negative 8 19 7

Total 237 194 81

APa 96.62% 90.21% 91.36%

TPa 98.12% 91.47% 92.67%

AP, apparent prevalence; TP, true prevalence.

ing T. buffeli/orientalis/sergenti complex (Schnittger et al., 2003). These parasites have a strikingly different feature of exhibiting a less marked leucocytic phase and not being able to transform their host cells in contrast with transforming Theileria species, T. parva, T. annulata and T. lestoquardi (Ahmed et al., 2006). Although T. uilenbergi-infected animals often die before piroplasms could be observed in blood smears and the schizonts could be demonstrated in many organs of infected animals (Yin et al., 2003), many surviving animals exhibit remarked anemia and icterus (Luo and Yin, 1997). It is thus assumed that not only schizonts but also merozoites of T. uilenbergi and T. luwenshuni play a pivotal role in the pathogenesis of the disease. Therefore, identication of unique and/or antigenic genes expressed at the merozoite stage may contribute

to the goal of being able to devise control strategies for this disease. In this study, we have identied an antigenic gene (TuIP) of T. uilenbergi from a merozoite cDNA library through immunoscreening. Bioinformatic analysis of TuIP found MHC I binding epitopes and 27 antigenic determinants, indicating its potential antigenicity. A BLASTp search through the available protozoan parasite genome in GenBank revealed that the TuIP is most closely related to two hypothetical proteins of T. parva and T. annulata with 32% and 31% identity, respectively. Interestingly, most known heterologous genes of T. uilenbergi to other Theileria species have exhibited high identities. These include TcD, showing 88% identity to a putative T. annulata membrane protein (TaD), TcSP partial cDNA, showing 94% identity to T. annulata surface protein (TaSP), TcSE partial cDNA, showing 99% identity to a secretory protein of T. annulata (TaSE) and Tc Clone-5, showing 100% identity to T. lestoquardi Clone-5 on the genomic level (Miranda et al., 2006b). Additionally, TcHSP70, the homologue of the T. lestoquardi HSP 70 obtained through immunoscreening, showed 95% identity to its heterologue in T. annulata and 97% identity in T. parva (Miranda et al., 2006a). The potential antigenicity of TuIP and its low identity to genes of other Theileria species has encouraged us to further characterise the TuIP recombinant protein and to develop a specic serological diagnostic test.

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In the present study, characterisation of the partially recombinantly expressed N-terminus of TuIP (rTuIP) on Western blot showed specic reaction with T. uilenbergi-positive sera. There was no cross-reaction with sera from animals infected with related pathogens, such as T. lestoquardi, Babesia sp. China or A. ovis. Interestingly, the mol. wt of rTuIP appeared higher (approximately 50 kDa) than predicted (41.7 kDa), most likely due to a high amount of dipeptide repeats containing proline. This is thought to result in an extended protein structure leading to a retarded migration during electrophoresis (Brewer et al., 1990). Similar phenomena have been observed with other Theileria proteins (Schnittger et al., 2002; Schneider et al., 2004). Additionally observed bands were also specically recognised by the anti-His-antibody, indicating that these are most likely breakdown fragments of the rTuIP protein during the protein expression and purication process. Blotted lysates of T. uilenbergi merozoites reacted with the rabbit anti-rTuIP anti-serum, leading to the identication of the native form of TuIP, which was a single protein band with a molecular size around 72 kDa. Previous studies on the identication of the blotted merozoite lysates using Theileria-positive sera demonstrated a spectrum of positively reacting merozoite antigens with different sizes, in which an approximate 71 kDa band was observed in Gannan, Longde and Zhangjiachuan isolates, but not in the Madang isolate (Gao, 2001). Later, the phylogenetic studies revealed that the isolates from Gannan, Longde and Zhangjiachuan belong to T. uilenbergi, while the Madang isolate belongs to T. luwenshuni (Schnittger et al., 2003; Yin et al., 2004), implying that the native TuIP is T. uilenbergi-specic and thus has the potential to be used for establishing species-specic diagnostic assays. The prospective use of rTuIP in specically detecting T. uilenbergi infection was veried by the observation that the rTuIP ELISA could only detect an antibody response in four out of six animals infected with T. luwenshuni with a low antibody titer. In contrast, when detecting T. uilenbergi infections, all animals showed strong antibody reactivity. These ndings again indicated that TuIP is very likely a T. uilenbergi merozoite-specic antigen, and may not be present in T. luwenshuni, since these parasites differ genetically, as has been demonstrated in phylogenetic analyses (Yin et al., 2004). The ambiguous results that rTuIP reacted with some positive sera of T. luwenshuni could be due to the fact that the partially recombinant expressed TuIP might not completely exclude shared epitopes with a similar antigen present in T. luwenshuni. However, this assumption should be dealt with in future studies. The partially recombinant expressed TuIP was used to develop an indirect ELISA in this study. The cut-off value of the rTuIP ELISA was determined to be 32.9 PP by testing 329 negative serum samples since large sample size can minimise the stochastic uncertainty in the cut-off selection (Greiner et al., 1994). Furthermore, through testing 128 reference positive and 48 negative samples, the sensitivity and specicity of the rTuIP ELISA were calculated to be 98.44% and 97.92%, respectively. This underlines the suitability of the rTuIP ELISA and its established cut-off. Additionally, the performance of the rTuIP ELISA was monitored by an internal quality control method, showing that it was stable and repeatable on a day to day basis. The rTuIP ELISA was applied to detect the antibody response of three blood-infected sheep and three sheep infected through tick infestation with T. uilenbergi. The results showed that antibodies could generally be detected earlier in sera of blood-infected sheep than in animals infected with tick infestation. In both cases, the antibodies could be detected for more than 3 months after infection. The previous study of these animals had shown that piroplasms in the blood-infected and the tick-infested sheep could be observed at 6 and 13 days p.i., respectively. In addition, parasitemia was low in both groups until day 27 and disease monitoring

showed signicantly elevated rectal temperature in the tick-infested animals compared with the blood-infected animals (Seitzer et al., 2008). In monitoring the antibody responses of these animals in this study, it was demonstrated that the tick-infested animals showed sharply increasing and stronger antibody responses compared with that of blood-infected animals. Tick infestation exposes all stages of the parasites life cycle to the host immune system, thus inducing a stronger immune response to the infection which is indicated by higher febrile (Seitzer et al., 2008) and/or by stronger antibody response in this study. Thus, the detection of the antibody response to the rTuIP ELISA reects the status of T. uilenbergi infection. Furthermore the potential usefulness and applicability of this ELISA method for detection of early infection and monitoring of persistent infection is demonstrated. Currently, a crude merozoite antigen-based ELISA is the only serological test applied for gathering the epidemiological data of theileriosis of small ruminants in China (Guo et al., 2007). However, the shortcomings of the crude antigen ELISA are difculties to standardise, to prepare the antigen and to exclude the crossreactivities to other related pathogens. In this study, the rTuIP ELISA was established using a recombinant protein originating from T. uilenbergi merozoites as a source. The test has been proven to be a suitable serological diagnostic tool for investigating the prevalence of Chinese theileriosis of small ruminants and could be an alternative serological method to the crude antigen ELISA in the future. However, due to limited information about the TuIP gene, future studies are aimed at the molecular characterisation of this gene and improving the specicity of detecting T. uilenbergi infection. Moreover, validation of the rTuIP ELISA with multi-methods and large sample sizes is yet to be performed. Acknowledgements Zhijie Liu is the recipient of a Deutscher Akademischer Austauschdienst (DAAD) scholarship. We would like to thank Mrs. Marisa Boettger for assistance in the purication of the rTuIP protein. This study was supported in part by the European Union (EU)-funded Coordinated Action Integrated Consortium on Ticks and Tick-borne Diseases (ICTTD-3), project number 510561, and Chinese projects: 863 (2006AA10A207), Supporting Plan (2007BAD40B00), National Natural Sciences Foundation (30800820; 30571397), the National Natural Resource Platform Project (2005DKA21100), Specic Fund for Sino-Europe Cooperation, Ministry of Science and Technology (MOST); Key Project of Gansu Province (0801NKDA033), Lanzhou, Gansu. State Key Laboratory of Veterinary Etiological Biology Project SKLVEB 2008ZZKT019 and National Public Interests Research Institute Basic Scientic Research Expenses Special Fund. References
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