International Journal for Parasitology 40 (2010) 591–598

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International Journal for Parasitology

journal homepage: www.elsevier.com/locate/ijpara

Identification 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,*
a
Division of Veterinary Infection Biology and Immunology, Research Center Borstel, Parkallee 22, D-23845 Borstel, Germany
b
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

a r t i c l e i n f o a b s t r a c t

Article history: Theileriosis of small ruminants in the northwest of China is a protozoan disease that restricts the devel-
Received 11 September 2009 opment of the livestock industry. The disease is caused by infection with Theileria uilenbergi and Thei-
Received in revised form 19 October 2009 leria luwenshuni, both of which are transmitted by ixodid Heamaphysalis ticks. The development of
Accepted 20 October 2009
serological tools as a means of integrated control of the disease is an urgent and important requirement.
Here we describe the identification and partial recombinant expression of a T. uilenbergi immunodomi-
nant protein (TuIP), which was identified by immunoscreening of a merozoite cDNA library. Using the
Keywords:
recombinant TuIP (rTuIP), a novel indirect ELISA was established using 329 negative serum samples to
ELISA
Immunodominant protein
determine the cut-off value. The internal quality control revealed satisfactory stability and repeatability
Theileria uilenbergi of the assay. Preliminary validation using 128 positive and 48 negative reference samples demonstrated
Theileriosis that the rTuIP ELISA is able to detect T. uilenbergi infection with high sensitivity and specificity. No cross-
reactivity was found in sera from animals infected with Theileria lestoquardi, Babesia sp. China or Ana-
plasma 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 specific antibodies against TuIP while
inoculation with infected blood induced an earlier production of TuIP-specific antibodies. The persistence
of the TuIP-specific 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.
Ó 2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

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 Haema-
physalis 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. luwensh-
uni 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 sta-
tus, 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 amplification (LAMP) (Liu et al., 2008b)
have been developed. Although highly sensitive and specific, RLB
and PCR are laborious and expensive, whereas LAMP as a novel
developed method still needs to be validated. In contrast, serolog-
ical 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 Ó 2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ijpara.2009.10.011
592 Z. Liu et al. / International Journal for Parasitology 40 (2010) 591–598
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 difficult to stan-
dardise, 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 cross-
reactivity 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-posi-
tive sera with the aim of identifying antigenic and specific 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 appli-
cability 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 Chi-
na and experimental sheep were purchased from a Theileria- and
other protozoa-free area. Blood films made from a drop of blood
from the ear vein were Giemsa-stained and microscopically exam-
ined prior to experimental infection. Only sheep negative for hae-
moparasites were used for producing negative serum samples
(sample No. 2201 and a further 329 negative samples collected
during the years 2003–2008). Additional negative sera were col-
lected from a slaughterhouse in Germany.
Animal experiments with sheep in China and rabbits in Ger-
many 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 field 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 Gan-
su 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. Identification of the TuIP gene
The construction of a cDNA library of T. uilenbergi from the mer-
ozoite 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 first 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 nitro-
cellulose membranes soaked in 10 mM isopropyl b-D-1-thiogalac-
topyranoside (IPTG) and dried on the surface of the plaques.
Subsequently, membranes were washed in PBS and the non-spe-
cific binding sites blocked in PBS, 2.5% fish gelatin (Sigma, Deis-
enhofen, Germany). Serum was diluted 1:500 in PBS with 0.1%
Tween 20 (PBST) containing 1% fish gelatin. Thereafter, filters 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, Ger-
many) diluted 1:20,000 in PBST, 1% fish gelatin was incubated
and filters then washed again as described above. Colorimetric
development was conducted via incubation of the membranes in
a substrate buffer that contained (Nitro-Blue Tetrazolium Chlo-
ride/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 sequenc-
ing (Eurofins MWG Operon, Ebersberg, Germany) using vector-spe-
cific primers (T3/T7, Stratagene, La Jolla, CA, USA).
2.3. Preparation of rTuIP
The N-terminus of the identified gene sequence of 2,181 bp con-
tained a unique BamHI restriction site from the kZAP vector se-
quence and a unique KpnI restriction site at nucleotides (nt)
1,067–1,072; these restriction sites were used to sub-clone the
N-terminal part of the gene into the pQE31 expression vector (Qia-
gen, Hilden, Germany), resulting in a recombinantly expressed pro-
tein 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 intro-
duced into the sequence by PCR amplification. 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 vec-
tor-specific primers (Qiagen, Hilden, Germany) (type III/IV forward
50 -CGGATAACAATTTCACACAG-30 and pQE reverse 50 -GTTCTGAGG
TCATTACTGG-30 ). Histidine tagged recombinant protein was ex-
pressed in M15 E. coli and purified using Ni–NTA agarose beads un-
der native conditions following the QiaExpressionist protocols
(Qiagen, Hilden, Germany). This first round of purified recombi-
nant protein was applied to re-purification using the Äkta prime
high system (Amersham Bioscience, Uppsala, Sweden) using 5 ml
HiTrap columns packed with Ni Sepharose 6 Fast Flow. The Ni-
chromatography was carried out according to the manufacturer’s
instructions using the following chromatography programme: (i)
washing of the column with 5–10 column volume (cv) H2O with
a flow rate of 5 ml/min; (ii) equilibration with 10 cv application
buffer (20 mM Tris–HCl, pH 8.0, 8 M urea, 0.5 M NaCl, 5 mM imid-
azole); (iii) application of the sample to the column with a flowrate
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 flow 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 gra-
dient of 500 mM to 1 M imidazole until the protein peak appears at
a flow 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 con-
centration of 0.2 mg/ml. This re-purified protein was used in all
experiments with Western blot and ELISA. Recombinant protein
expression was verified in Western blots using an anti-histidine
antibody and T. uilenbergi-positive serum samples. Purity was as-
 Z. Liu et al. / International Journal for Parasitology 40 (2010) 591–598
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 purified 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-immu-
nized 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 purified rTuIP protein at 2 week intervals, whereby
the first inoculation was conducted with FCA and the following
three boosts with incomplete Freund’s adjuvant. The successful
production of the anti-TuIP anti-serum was verified by dot blot
detection prior to animal bleeding. The rabbit was bled 10 days
after the final 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 SDS–PAGE using 12% polyacrylamide gels under reduc-
ing conditions and transferred to nitrocellulose membranes. To ver-
ify the expression and purification 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 pro-
teins. 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 second-
ary antibody for these experiments was AP-conjugated goat anti-
rabbit immunoglobulin antibody (1:2,500; Dako, Glostrup, Den-
mark). 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 con-
jugate (peroxidase-conjugated Affinipure 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 posi-
tive and negative sera. Thereafter, a standard indirect ELISA proto-
col was established.
Coating was done by adding 100 ll antigen (TuIP) solution
(0.5 lg/ml in carbonate bicarbonate buffer (pH 9.6) (Sigma, Deis-
enhofen, Germany)) into each well of 96-well ELISA plates (Maxi-
sorp, Nunc, Glostrup, Denmark) except for four blank control
wells filled 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
593
Friedrichshall, Germany). Non-specific binding sites in each well
were blocked using 200 ll 1% (w/v) BSA (Sigma, Deisenhofen, Ger-
many) 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 test-
ing. Control positive (C+) and control negative (C) sera were ap-
plied 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 con-
trols)]  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 field samples from three locations as described
above was examined using the established ELISA. Based on the cal-
culated specificity (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 sam-
ples from both tests  100) of the rTuIP ELISA, the apparent preva-
lence (AP) and true prevalence (TP) were calculated, respectively,
using the following formula:
AP ¼ number of positive samples=number of total samples
TP ¼ ðAP þ Sp  1Þ=ðSe þ Sp  1Þ
3. Results
3.1. Identification 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
significant 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, consist-
ing of 27 antigenic determinants (Kolaskar and Tongaonkar, 1990).
594 Z. Liu et al. / International Journal for Parasitology 40 (2010) 591–598

Fig. 2. Specificity and testing of cross-reactivity of the recombinant Theileria uilen-

bergi immunodominant protein (TuIP) using ELISA. Sera 1–6, T. uilenbergi-positive
Fig. 1. Specificity and testing of cross-reactivity of the recombinant Theileria uilen-
serum samples Nos. 1201, 1203, 1205, 1410, 1411, 1412; sera 7, T. lestoquardi-
bergi immunodominant protein (TuIP) using Western blot. Lane M, mol. wt marker;
positive serum Tl17; sera 8, Babesia sp. China-positive serum; sera 9, Anaplama ovis-
lanes 1–6, T. uilenbergi-positive serum samples Nos. 1201, 1203, 1205, 1410, 1411,
positive serum; sera 10 and 11, negative serum. Each sample was diluted 1:200,
1412; lane 7, T. lestoquardi-positive serum Tl17; lane 8, Babesia sp. China-positive
1:400 and 1:800. The cut-off of the ELISA is indicated by a horizontal line. As in the
serum; lane 9, Anaplasma ovis-positive serum; lanes 10 and 11, negative serum;
Western blot (Fig. 1), only sera from Theileria (China)-infected animals show
lane 12, donkey anti-sheep secondary antibody; lane 13, mouse anti-RGS-His-
binding of specific antibodies resulting at a signal above the established threshold.
antibody; lane 14, goat anti-mouse secondary antibody control. A specific band at
approximately 50 kD was detected by the T. uilenbergi-positive serum samples and
the anti-His-antibody, together with specific smaller bands most likely correspond-
ing to breakdown products of the protein. No protein bands were detected with
serum from animals infected with other small ruminant tick-borne pathogens.

Analysis of major histocompatibility complex I (MHC-I) binding

epitopes based on bovine class I A20 specificity (http://www-bi-
mas.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 identified gene encoded for a potentially antigenic pro-
tein 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-ter-
minal 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
Fig. 3. The lysate of purified Theileria uilenbergi merozoites was blotted and tested
cross-reactivity was seen with serum samples of T. lestoquardi-,
for reactivity with rabbit anti-recombinant T. uilenbergi immunodominant protein
Babesia sp. China- and A. ovis-infected animals or negative sera (rTuIP) antibody. Lane M, mol. wt marker; lanes 1 and 2, 1:2,000 and 1:4,000 diluted
using both Western blots (Fig. 1) and ELISA (Fig. 2). This indicated rabbit anti-rTuIP anti-serum; lanes 3 and 4, 1:2,000 and 1:4,000 diluted rabbit anti-
that the TuIP gene encodes a potential immunodominant protein of TuIP anti-serum after blocking by 3.5 lg rTuIP; lanes 5 and 6, 1:2,000 and 1:4,000
T. uilenbergi and it was thus termed T. uilenbergi Immunodominant diluted pre-immunized rabbit serum; lane 7, goat anti-rabbit secondary antibody
control.
Protein (TuIP) (Genbank Accession No. FJ467922).
Immunization of a rabbit with rTuIP resulted in a specific anti-
rTuIP anti-serum with a titer of 1:51,200 (data not shown). This positive and negative samples, these conditions were used in all
anti-serum was used to identify the native TuIP protein in merozo- subsequent experiments (data not shown).
ite crude antigen of T. uilenbergi in Western blots. The results The internal quality control was assessed by testing positive
showed that the rabbit anti-TuIP anti-serum could specifically rec- sample 1229P and negative sample 1229N in six different plates
ognise a single parasite protein band around 72 kDa in size (Fig. 3). on two different days (Table 1). Similar values for the samples were
Pre-absorption of the rabbit anti-rTuIP anti-serum with 3.5 lg of
rTuIP protein inhibited the detection of this protein band, indicat-
Table 1
ing the specificity of the reaction. Moreover, the pre-immunization
Internal quality control results from the recombinant Theileria uilenbergi immuno-
rabbit serum was also negative. These data further confirmed the dominant protein (rTuIP) indirect ELISA by measuring six different plates on two
parasite origin and the immunodominant feature of the TuIP different days.
protein.
Plate number C++ PP C PP CC PP
Day 1 1 108.05 ± 3.41 3.88 ± 0.25 -0.19 ± 0.07
3.2. Establishment of the rTuIP indirect ELISA 2 94.17 ± 2.04 3.10 ± 0.24 -0.24 ± 0.16
3 104.25 ± 3.91 4.34 ± 0.28 -0.15 ± 0.22
The optimal dilutions of serum samples and conjugate were Day 2 1 97.19 ± 1.34 3.15 ± 0.43 -0.08 ± 0.18
checkerboard-titrated using known positive and negative serum 2 104.31 ± 2.61 4.27 ± 0.49 -0.16 ± 0.20
samples diluted twofold from 1:100 to 1:3,200, while conjugate 3 107.43 ± 5.60 4.24 ± 0.33 -0.04 ± 0.10
was diluted at 1:10,000, 1:12,500, 1:15,000 and 1:20,000. Since C++ PP, mean percent positivity (PP) value of the positive control; C PP, mean PP
the serum sample dilution at 1:400 and conjugate dilution at value of negative control; CC PP, mean PP value of conjugate control.
1:15,000 gave the maximum contrast in the O.D. value between Average values of four tests are given with S.D.
 Z. Liu et al. / International Journal for Parasitology 40 (2010) 591–598
Fig. 4. Preliminary validation of the recombinant Theileria uilenbergi immunodom-
inant 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 defined as the mean PP value
of 329 negative serum samples plus two times the S.D. Before sam-
pling, blood smears of all animals were examined under the micro-
scope, confirming the absence of haemoparasites. Moreover,
subsequent observation of these animals gave no indication that
they were infected by protozoa before transportation to the labora-
tory. 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 previ-
ous investigations on sera collected from the field using crude anti-
gen 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 ref-
erence samples were negative, resulting in a calculated sensitivity
and specificity of 98.44% and 97.92%, respectively (Fig. 4 and
Table 2).
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. luwensh-
uni-infected animals against rTuIP protein was separately investi-
gated using the rTuIP ELISA. Six experimental sheep were
Table 2
Preliminary validation of the sensitivity and specificity of the recombinant Theileria
uilenbergi immunodominant protein (rTuIP) ELISA using positive and negative
reference serum samples.
Crude antigen ELISA
Positive Negative Total
rTuIP ELISA Positive 126 1 127
Negative 2 47 49
Total 128 48 176
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).
Specificity 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).
595
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.
infected by blood inoculation with the Weiyuan strain verified by
PCR to be T. luwenshuni (Yin et al., 2008). Serum samples were col-
lected at days 0, 27 and 47 post inoculation. The successful infec-
tion 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. uilen-
bergi-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 in-
fected with T. uilenbergi against rTuIP, the serum samples from dif-
ferent 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 Lin-
tan strain, and another three (Nos. 1250, 1240 and 1229) were in-
fected by feeding 200 H. qinghaiensis ticks from an endemic region
(Seitzer et al., 2008). Infection of the animals was verified accord-
ing to the observation of parasites in blood smears. The results of
the ELISA demonstrated that the infection with infected blood in-
duced antibodies binding to rTuIP in less than 14 days and in-
creased 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 ani-
mals, in both infection models the rTuIP ELISA was able to detect
specific antibodies more than 3 months p.i. This indicates that
the test is a suitable tool for detection of early infection and mon-
itoring of persistent infections with T. uilenbergi (Fig. 6).
3.4. Detection of circulating anti-rTuIP antibodies in field samples from
theileriosis endemic regions
The field 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 respec-
tive 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-
596 Z. Liu et al. / International Journal for Parasitology 40 (2010) 591–598

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 significant 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 to the goal of being able to devise control strategies for this disease.
Detection of specific antibodies in 512 field serum samples from China, using the In this study, we have identified an antigenic gene (TuIP) of T. uilen-
recombinant Theileria uilenbergi immunodominant protein (rTuIP) ELISA.
bergi from a merozoite cDNA library through immunoscreening.
Locations Positive Negative Total APa TPa Bioinformatic analysis of TuIP found MHC I binding epitopes and
Azitan 229 8 237 96.62% 98.12% 27 antigenic determinants, indicating its potential antigenicity. A
Ganjia 175 19 194 90.21% 91.47% BLASTp search through the available protozoan parasite genome
Yangyong 74 7 81 91.36% 92.67% in GenBank revealed that the TuIP is most closely related to two
a
AP, apparent prevalence; TP, true prevalence. 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
ing’ T. buffeli/orientalis/sergenti complex (Schnittger et al., 2003). identities. These include TcD, showing 88% identity to a putative T.
These parasites have a strikingly different feature of exhibiting a annulata membrane protein (TaD), TcSP partial cDNA, showing 94%
less marked leucocytic phase and not being able to transform their identity to T. annulata surface protein (TaSP), TcSE partial cDNA,
host cells in contrast with transforming Theileria species, T. parva, showing 99% identity to a secretory protein of T. annulata (TaSE)
T. annulata and T. lestoquardi (Ahmed et al., 2006). Although T. and Tc Clone-5, showing 100% identity to T. lestoquardi Clone-5 on
uilenbergi-infected animals often die before piroplasms could be the genomic level (Miranda et al., 2006b). Additionally, TcHSP70,
observed in blood smears and the schizonts could be demonstrated the homologue of the T. lestoquardi HSP 70 obtained through
in many organs of infected animals (Yin et al., 2003), many surviv- immunoscreening, showed 95% identity to its heterologue in T.
ing animals exhibit remarked anemia and icterus (Luo and Yin, annulata and 97% identity in T. parva (Miranda et al., 2006a). The
1997). It is thus assumed that not only schizonts but also merozo- potential antigenicity of TuIP and its low identity to genes of other
ites of T. uilenbergi and T. luwenshuni play a pivotal role in the path- Theileria species has encouraged us to further characterise the TuIP
ogenesis of the disease. Therefore, identification of unique and/or recombinant protein and to develop a specific serological diagnos-
antigenic genes expressed at the merozoite stage may contribute tic test.
 Z. Liu et al. / International Journal for Parasitology 40 (2010) 591–598
In the present study, characterisation of the partially recombi-
nantly expressed N-terminus of TuIP (rTuIP) on Western blot
showed specific 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. Inter-
estingly, 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 phe-
nomena have been observed with other Theileria proteins (Schnitt-
ger et al., 2002; Schneider et al., 2004). Additionally observed
bands were also specifically recognised by the anti-His-antibody,
indicating that these are most likely breakdown fragments of the
rTuIP protein during the protein expression and purification
process.
Blotted lysates of T. uilenbergi merozoites reacted with the rab-
bit anti-rTuIP anti-serum, leading to the identification of the native
form of TuIP, which was a single protein band with a molecular size
around 72 kDa. Previous studies on the identification 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 Gan-
nan, Longde and Zhangjiachuan isolates, but not in the Madang iso-
late (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-specific and thus has the potential to be used
for establishing species-specific diagnostic assays.
The prospective use of rTuIP in specifically detecting T. uilen-
bergi infection was verified 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 findings again indicated that TuIP is very
likely a T. uilenbergi merozoite-specific 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 posi-
tive 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 sam-
ples since large sample size can minimise the stochastic uncer-
tainty in the cut-off selection (Greiner et al., 1994). Furthermore,
through testing 128 reference positive and 48 negative samples,
the sensitivity and specificity of the rTuIP ELISA were calculated
to be 98.44% and 97.92%, respectively. This underlines the suitabil-
ity of the rTuIP ELISA and its established cut-off. Additionally, the
performance of the rTuIP ELISA was monitored by an internal qual-
ity 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 infec-
tion. The previous study of these animals had shown that piro-
plasms in the blood-infected and the tick-infested sheep could be
observed at 6 and 13 days p.i., respectively. In addition, parasite-
mia was low in both groups until day 27 and disease monitoring
597
showed significantly elevated rectal temperature in the tick-in-
fested 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 com-
pared with that of blood-infected animals. Tick infestation exposes
all stages of the parasite’s 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 stron-
ger antibody response in this study. Thus, the detection of the anti-
body response to the rTuIP ELISA reflects 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). How-
ever, the shortcomings of the crude antigen ELISA are difficulties
to standardise, to prepare the antigen and to exclude the cross-
reactivities to other related pathogens. In this study, the rTuIP ELI-
SA 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 alterna-
tive 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 specificity 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 Aust-
auschdienst (DAAD) scholarship. We would like to thank Mrs. Marisa
Boettger for assistance in the purification 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), Specific 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 Insti-
tute Basic Scientific Research Expenses Special Fund.
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