Documentos de Académico
Documentos de Profesional
Documentos de Cultura
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 Theileria sergenti groups (Schnittger et al., 2003; Yin et al., 2004).
It is assumed that these closely related parasites share the common
Theileriosis of small ruminants caused by Theileria uilenbergi feature of exhibiting a less marked leucocytic phase and not being
and Theileria luwenshuni (Yin et al., 2007) is a severe and often able to transform their host cells (Ahmed et al., 2006).
lethal disease, constituting a severe restriction for the development Regarding the diagnosis of the disease, traditionally T. luwensh-
of the small ruminant livestock industry in the northwest of China, uni and T. uilenbergi infections are examined by observation of
especially regarding the use of exotic animals (Luo and Yin, 1997). merozoites in Giemsa-stained blood smears under the microscope
Ixodid Haemaphysalis ticks, Haemaphysalis qinghaiensis and Haema- and/or observation of the clinical signs of the infected animals (Luo
physalis longicornis have been demonstrated to be responsible for and Yin, 1997). However, these methods do not detect carrier sta-
transmission of the disease (Yin et al., 2002; Li et al., 2007, tus, are time consuming, not very sensitive and require operators
2009). Although a recent report of a protozoan isolated in Spain with high expertise. To improve diagnostic methods, PCR (Yin
had molecular similarity at the 18S rRNA gene level to Chinese et al., 2008), reverse line blot (RLB) (Schnittger et al., 2004) and
ovine Theileria (Nagore et al., 2004), the only disease reports to date loop mediated isothermal amplification (LAMP) (Liu et al., 2008b)
are from China. Molecular phylogenetic studies have shown that have been developed. Although highly sensitive and specific, RLB
the Chinese Theileria is closely related to the Theileria buffeli and and PCR are laborious and expensive, whereas LAMP as a novel
developed method still needs to be validated. In contrast, serolog-
q
ical diagnostic tools are not only relatively inexpensive, practical,
Note: Nucleotide sequence data reported in this paper is available in the
GenBank database under the Accession No. FJ467922.
easy to use and suitable for large scale surveys, but are also an
* Corresponding author. Tel.: +49 4573 188 413; fax: +49 4537 188 627. important method for monitoring the antibody responses of
E-mail address: useitzer@fz-borstel.de (U. Seitzer). 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 (Schleicher and Schuell, Dassel, Germany). Plaques were generated
merozoite antigen (Gao et al., 2002), another based on the partially by plating the library onto Luria broth (LB) plates according to the
expressed Theileria lestoquardi recombinant heat shock protein 70 manual of the picoBlue™ immunoscreening kit (Stratagene, La Jolla,
(rTIHSP 70) (Miranda et al., 2006a). There is still, however, a need CA, USA). Phages were induced to express protein by placing nitro-
for improvement, as the crude antigen ELISA is difficult to stan- cellulose membranes soaked in 10 mM isopropyl b-D-1-thiogalac-
dardise, requires infection of animals for antigen preparation and topyranoside (IPTG) and dried on the surface of the plaques.
is potentially cross-reactive with other related pathogens. On the Subsequently, membranes were washed in PBS and the non-spe-
other hand, the rTIHSP 70 gene used for the recombinant protein cific binding sites blocked in PBS, 2.5% fish gelatin (Sigma, Deis-
ELISA originated from T. lestoquardi and bears the risk of cross- enhofen, Germany). Serum was diluted 1:500 in PBS with 0.1%
reactivity with other piroplasms infecting small ruminants. Tween 20 (PBST) containing 1% fish gelatin. Thereafter, filters were
In the present study, a T. uilenbergi merozoite cDNA library (Liu incubated with serum at room temperature for 2 h while shaking.
et al., 2008a) was immunoscreened with pooled T. uilenbergi-posi- After washing in PBST, a conjugate (donkey anti-sheep alkaline
tive sera with the aim of identifying antigenic and specific genes of phosphatase-conjugated Fab fragments; Dianova, Hamburg, Ger-
T. uilenbergi. An antigenic gene termed TuIP was obtained and a many) diluted 1:20,000 in PBST, 1% fish gelatin was incubated
partially expressed recombinant TuIP (rTuIP) was characterised and filters then washed again as described above. Colorimetric
and applied for establishment of a novel ELISA. The potential appli- development was conducted via incubation of the membranes in
cability of the ELISA was investigated by analysing serum from a substrate buffer that contained (Nitro-Blue Tetrazolium Chlo-
experimentally infected sheep and sera collected from endemic ride/5-Bromo-4-Chloro-30 -Indolyphosphate p-Toluidine salt (NBT/
regions. 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-
2. Materials and methods
cific primers (T3/T7, Stratagene, La Jolla, CA, USA).
sessed by Coomassie gel staining, and protein concentration was Friedrichshall, Germany). Non-specific binding sites in each well
determined using the BioRadMicro-DC Assay kit (BioRad, Munich, were blocked using 200 ll 1% (w/v) BSA (Sigma, Deisenhofen, Ger-
Germany). 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
2.4. Preparation of rabbit anti-rTuIP anti-serum 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-
The purified TuIP recombinant protein was used to immunize plied in four replicates and four wells were always used as a
three rabbits for generation of rabbit anti-rTuIP anti-serum conjugate control (CC), receiving only the diluent buffer. Plates
(Charles River, GmbH, Kisslegg, Germany). Sera of three pre-immu- were incubated for 1 h at room temperature on an orbital shaker,
nized rabbits were tested by Western blots of the rTuIP protein, followed by a washing step. Then 100 ll of the conjugate horse
and the one showing minimal background reaction was chosen radish peroxidase-labelled rabbit anti-sheep antibody (Dianova,
for immunization. The immunization regime covered four boosts Hamburg, Germany) diluted 1:15,000 in the diluent buffer were
each of 200 lg purified rTuIP protein at 2 week intervals, whereby added to all wells and incubated. After washing, 100 ll of freshly
the first inoculation was conducted with FCA and the following prepared substrate solution [H2O2/tetramethylbenzi-dine (TMB,
three boosts with incomplete Freund’s adjuvant. The successful Sigma, Deisenhofen, Germany) in citric acid buffer (pH 4.0)] were
production of the anti-TuIP anti-serum was verified by dot blot dispensed into each well. Colour development was in the dark
detection prior to animal bleeding. The rabbit was bled 10 days for 10 min and the reaction stopped by adding 100 ll 1 N H2SO4.
after the final boost and the serum stored at 20 °C until used. The absorbance at 450 nm was measured using an ELISA reader
(Expert 96, Asys Hightech, Overath, Austria; and Stat Fax 2100,
2.5. Western blot analysis Awareness Technology, Palm City, FL, USA).
The obtained O.D. values were expressed as percent positivity
The preparation of merozoite crude antigen was carried out (PP) of the internal positive control as follows: [(average O.D. value
according to the protocol described by Gao et al. (2002). Optimal of testing samples average O.D. value of blank controls)/(average
amounts of merozoite crude antigen and recombinant proteins were O.D. value of the positive controls average O.D. value of blank con-
subjected to SDS–PAGE using 12% polyacrylamide gels under reduc- trols)] 100. The ELISA cut-off value, which served as the threshold
ing conditions and transferred to nitrocellulose membranes. To ver- between positive and negative sera, was determined as the mean PP
ify the expression and purification of the recombinant proteins, the value obtained from the 329 negative serum samples plus 2 S.D.
RGS-His™ mouse anti-histidine antibody (1:4,000, Qiagen, Hilden,
Germany) and secondary alkaline phosphatase (AP) conjugated goat 2.7. Application of the rTuIP indirect ELISA
anti-mouse IgG + IgM (H+L) antibody (1:10,000, Dianova, Hamburg,
Germany) were used to detect the His-tag on the recombinant pro- A total of 512 field samples from three locations as described
teins. Merozoite crude antigen and rTuIP were Western blotted above was examined using the established ELISA. Based on the cal-
and probed with pre-immunization rabbit serum, rabbit anti-rTuIP culated specificity (Sp = #samples negative in both tests/total
anti-serum and rabbit anti-rTuIP anti-serum pre-absorbed with number of negative samples from both tests 100) and sensitivity
rTuIP (1:2,000 and 1:4,000) for detection of the protein. The second- (Se = #samples positive in both tests/total number of positive sam-
ary antibody for these experiments was AP-conjugated goat anti- ples from both tests 100) of the rTuIP ELISA, the apparent preva-
rabbit immunoglobulin antibody (1:2,500; Dako, Glostrup, Den- lence (AP) and true prevalence (TP) were calculated, respectively,
mark). All of the serum samples and the secondary antibodies were using the following formula:
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). AP ¼ number of positive samples=number of total samples
Binding of secondary antibody was detected with a BCIP/NBT TP ¼ ðAP þ Sp 1Þ=ðSe þ Sp 1Þ
substrate.
Serum 1229P, collected after observation of the piroplasms in 3.1. Identification of TuIP
Giemsa-stained blood smear at day 58 post inoculation in an
experimentally infected sheep, served as the positive control. Sequence analysis of the clone isolated by immunoscreening of
Negative control serum sample 1229N was collected from the the merozoite library revealed an incomplete open reading frame
same animal prior to infection. The optimum dilutions of the rTuIP (ORF) lacking a start codon with a size of 2,181 bp. This incomplete
antigen, positive serum 1229P, negative serum 1229N and the con- ORF encoded for a protein of 670 amino acids with a predicted mol.
jugate (peroxidase-conjugated Affinipure rabbit anti-sheep IgG wt of 74.8 kDa. In addition, the clone contained 167 bp of the 30
(H+L, Dianova, Hamburg, Germany)) were checkerboard-titrated untranslated region with 37 bp of a poly A tail. BLASTn searches
on a 96-well ELISA plate. The optimum dilution considered was within available Apicomplexa genome databases (http://www.
the highest dilution of antigen/conjugate that still saturated the ncbi.nlm.nih.gov/sutils/genom_tree.cgi?organism=euk) found no
plate and gave maximum contrast in terms of O.D. between posi- significant similarity, while BLASTp searches showed the highest
tive and negative sera. Thereafter, a standard indirect ELISA proto- identity (Identities = 218/678 (32%), Positives = 331/678 (48%),
col was established. Gaps = 55/678 (8%) to a hypothetical protein of Theileria parva
Coating was done by adding 100 ll antigen (TuIP) solution (Gene ID: 3503410 TP01_0987) and its Theileria annulata
(0.5 lg/ml in carbonate bicarbonate buffer (pH 9.6) (Sigma, Deis- orthologue (Identities = 216/681 (31%), Positives = 327/681 (48%),
enhofen, Germany)) into each well of 96-well ELISA plates (Maxi- Gaps = 80/681 (11%), polymorphic antigen precursor-like protein,
sorp, Nunc, Glostrup, Denmark) except for four blank control Gene ID: 3864034 TA16685) (Supplementary online material in
wells filled with carbonate bicarbonate buffer only. The plates Pain et al., 2005). Using a prediction server for antigenicity
were incubated at 4 °C overnight, then washed with PBS containing (http://immunax.dfci.harvard.edu/Tools/antigenic.pl) the average
0.05% (v/v) Tween 20 (pH 7.4) in three dispensing/aspiration cycles antigenic propensity for this protein was given as 1.0253, consist-
using a Bio-Tek 405 automated ELISA plate washer (Bio-Tek, Bad ing of 27 antigenic determinants (Kolaskar and Tongaonkar, 1990).
594 Z. Liu et al. / International Journal for Parasitology 40 (2010) 591–598
Sensitivity of the rTuIP ELISA = 98.43% (number of samples positive in both tests/
4. Discussion
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/ Previous phylogenetic studies on T. uilenbergi and T. luwenshuni
total number of negative samples from both tests 100 = 47/48 100). 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) 591–598 597
In the present study, characterisation of the partially recombi- showed significantly elevated rectal temperature in the tick-in-
nantly expressed N-terminus of TuIP (rTuIP) on Western blot fested animals compared with the blood-infected animals (Seitzer
showed specific reaction with T. uilenbergi-positive sera. There et al., 2008). In monitoring the antibody responses of these animals
was no cross-reaction with sera from animals infected with related in this study, it was demonstrated that the tick-infested animals
pathogens, such as T. lestoquardi, Babesia sp. China or A. ovis. Inter- showed sharply increasing and stronger antibody responses com-
estingly, the mol. wt of rTuIP appeared higher (approximately pared with that of blood-infected animals. Tick infestation exposes
50 kDa) than predicted (41.7 kDa), most likely due to a high all stages of the parasite’s life cycle to the host immune system,
amount of dipeptide repeats containing proline. This is thought thus inducing a stronger immune response to the infection which
to result in an extended protein structure leading to a retarded is indicated by higher febrile (Seitzer et al., 2008) and/or by stron-
migration during electrophoresis (Brewer et al., 1990). Similar phe- ger antibody response in this study. Thus, the detection of the anti-
nomena have been observed with other Theileria proteins (Schnitt- body response to the rTuIP ELISA reflects the status of T. uilenbergi
ger et al., 2002; Schneider et al., 2004). Additionally observed infection. Furthermore the potential usefulness and applicability of
bands were also specifically recognised by the anti-His-antibody, this ELISA method for detection of early infection and monitoring
indicating that these are most likely breakdown fragments of the of persistent infection is demonstrated.
rTuIP protein during the protein expression and purification Currently, a crude merozoite antigen-based ELISA is the only
process. serological test applied for gathering the epidemiological data of
Blotted lysates of T. uilenbergi merozoites reacted with the rab- theileriosis of small ruminants in China (Guo et al., 2007). How-
bit anti-rTuIP anti-serum, leading to the identification of the native ever, the shortcomings of the crude antigen ELISA are difficulties
form of TuIP, which was a single protein band with a molecular size to standardise, to prepare the antigen and to exclude the cross-
around 72 kDa. Previous studies on the identification of the blotted reactivities to other related pathogens. In this study, the rTuIP ELI-
merozoite lysates using Theileria-positive sera demonstrated a SA was established using a recombinant protein originating from T.
spectrum of positively reacting merozoite antigens with different uilenbergi merozoites as a source. The test has been proven to be a
sizes, in which an approximate 71 kDa band was observed in Gan- suitable serological diagnostic tool for investigating the prevalence
nan, Longde and Zhangjiachuan isolates, but not in the Madang iso- of Chinese theileriosis of small ruminants and could be an alterna-
late (Gao, 2001). Later, the phylogenetic studies revealed that the tive serological method to the crude antigen ELISA in the future.
isolates from Gannan, Longde and Zhangjiachuan belong to However, due to limited information about the TuIP gene, future
T. uilenbergi, while the Madang isolate belongs to T. luwenshuni studies are aimed at the molecular characterisation of this gene
(Schnittger et al., 2003; Yin et al., 2004), implying that the native and improving the specificity of detecting T. uilenbergi infection.
TuIP is T. uilenbergi-specific and thus has the potential to be used Moreover, validation of the rTuIP ELISA with multi-methods and
for establishing species-specific diagnostic assays. large sample sizes is yet to be performed.
The prospective use of rTuIP in specifically detecting T. uilen-
bergi infection was verified by the observation that the rTuIP ELISA Acknowledgements
could only detect an antibody response in four out of six animals
infected with T. luwenshuni with a low antibody titer. In contrast, Zhijie Liu is the recipient of a Deutscher Akademischer Aust-
when detecting T. uilenbergi infections, all animals showed strong auschdienst (DAAD) scholarship. We would like to thank Mrs. Marisa
antibody reactivity. These findings again indicated that TuIP is very Boettger for assistance in the purification of the rTuIP protein. This
likely a T. uilenbergi merozoite-specific antigen, and may not be study was supported in part by the European Union (EU)-funded
present in T. luwenshuni, since these parasites differ genetically, Coordinated Action ‘‘Integrated Consortium on Ticks and Tick-borne
as has been demonstrated in phylogenetic analyses (Yin et al., Diseases” (ICTTD-3), project number 510561, and Chinese projects:
2004). The ambiguous results that rTuIP reacted with some posi- ‘‘863” (2006AA10A207), Supporting Plan (2007BAD40B00), National
tive sera of T. luwenshuni could be due to the fact that the partially Natural Sciences Foundation (30800820; 30571397), the National
recombinant expressed TuIP might not completely exclude shared Natural Resource Platform Project (2005DKA21100), Specific Fund
epitopes with a similar antigen present in T. luwenshuni. However, for Sino-Europe Cooperation, Ministry of Science and Technology
this assumption should be dealt with in future studies. (MOST); Key Project of Gansu Province (0801NKDA033), Lanzhou,
The partially recombinant expressed TuIP was used to develop Gansu. State Key Laboratory of Veterinary Etiological Biology Project
an indirect ELISA in this study. The cut-off value of the rTuIP ELISA SKLVEB 2008ZZKT019 and National Public Interests Research Insti-
was determined to be 32.9 PP by testing 329 negative serum sam- tute Basic Scientific Research Expenses Special Fund.
ples since large sample size can minimise the stochastic uncer-
tainty in the cut-off selection (Greiner et al., 1994). Furthermore, References
through testing 128 reference positive and 48 negative samples,
the sensitivity and specificity of the rTuIP ELISA were calculated Ahmed, J.S., Luo, J., Schnittger, L., Seitzer, U., Jongejan, F., Yin, H., 2006. Phylogenetic
position of small-ruminant infecting piroplasms. Ann. NY Acad. Sci. 1081, 498–
to be 98.44% and 97.92%, respectively. This underlines the suitabil- 504.
ity of the rTuIP ELISA and its established cut-off. Additionally, the Bakheit, M.A., Seitzer, U., Ahmed, J.S., 2006. A new recombinant protein-based ELISA
performance of the rTuIP ELISA was monitored by an internal qual- for the diagnosis of malignant theileriosis of sheep and goats. Parasitol. Res. 98,
145–149.
ity control method, showing that it was stable and repeatable on a Brewer, S., Tolley, M., Trayer, I.P., Barr, G.C., Dorman, C.J., Hannavy, K., Higgins, C.F.,
day to day basis. Evans, J.S., Levine, B.A., Wormald, M.R., 1990. Structure and function of X-Pro
The rTuIP ELISA was applied to detect the antibody response of dipeptide repeats in the TonB proteins of Salmonella typhimurium and
Escherichia coli. J. Mol. Biol. 216, 883–895.
three blood-infected sheep and three sheep infected through tick Gao, Y.L., 2001. Studies on merozoite antigenic protein analysis of Chinese Theileria
infestation with T. uilenbergi. The results showed that antibodies isolates and establishment of diagnostic method and chemical treatment of the
could generally be detected earlier in sera of blood-infected sheep disease. Dissertation of Master Degree, P1-64. Lanzhou Veterinary Research
Institute, Lanzhou, China.
than in animals infected with tick infestation. In both cases, the
Gao, Y.L., Yin, H., Luo, J.X., Ouyang, W.Q., Bao, H.M., Guan, G.Q., Zhang, Q.C., Lu, W.S.,
antibodies could be detected for more than 3 months after infec- Ma, M.L., 2002. Development of an enzyme-linked immunosorbent assay for the
tion. The previous study of these animals had shown that piro- diagnosis of Theileria sp. infection in sheep. Parasitol. Res. 88, S8–S10.
plasms in the blood-infected and the tick-infested sheep could be Greiner, M., Franke, C.R., Bohning, D., Schlattmann, P., 1994. Construction of an
intrinsic cut-off value for the sero-epidemiological study of Trypanosoma evansi
observed at 6 and 13 days p.i., respectively. In addition, parasite- infections in a canine population in Brazil: a new approach towards an unbiased
mia was low in both groups until day 27 and disease monitoring estimation of prevalence. Acta Trop. 56, 97–109.
598 Z. Liu et al. / International Journal for Parasitology 40 (2010) 591–598
Guo, S., Mu, Y., Liu, Z., Ma, D., Yang, S., Ge, G., Fang, B., Ga, D., Ma, M., Luo, J., Yin, H., McKeever, Shiels, B., Tait, A., Barrell, B., Hall, N., 2005. Genome of the host–cell
Seitzer, U., Ahmed, J.S., 2007. Serological investigation of ovine theileriosis by transforming parasite Theileria annulata compared with T. parva. Science 309,
ELISA in Gannan Tibet Region of Gansu Province in China. Parasitol. Res. 101 131–133.
(Suppl. 2), S197–S200. Parker, K.C., Bednarek, M.A., Coligan, J.E., 1994. Scheme for ranking potential HLA-
Kolaskar, A.S., Tongaonkar, P.C., 1990. A semi-empirical method for prediction of A2 binding peptides based on independent binding of individual peptide side-
antigenic determinants on protein antigens. FEBS Lett. 276, 172–174. chains. J. Immunol. 152, 163–175.
Li, Y., Luo, J., Liu, Z., Guan, G., Gao, J., Ma, M., Dang, Z., Liu, A., Ren, Q., Lu, B., Liu, J., Schneider, I., Haller, D., Seitzer, U., Beyer, D., Ahmed, J.S., 2004. Molecular genetic
Zhao, H., Li, J., Liu, G., Bai, Q., Yin, H., 2007. Experimental transmission of characterization and subcellular localization of a putative Theileria annulata
Theileria sp. (China 1) infective for small ruminants by Haemaphysalis longicornis membrane protein. Parasitol. Res. 94, 405–415.
and Haemaphysalis qinghaiensis. Parasitol. Res. 101, 533–538. Schnittger, L., Katzer, F., Biermann, R., Shayan, P., Boguslawski, K., McKellar, S.,
Li, Y., Luo, J., Guan, G., Ma, M., Liu, A., Liu, J., Ren, Q., Niu, Q., Lu, B., Gao, J., Liu, Z., Dang, Z., Beyer, D., Shiels, B.R., Ahmed, J.S., 2002. Characterization of a polymorphic
Tian, Z., Zhang, B., He, Z., Bai, Q., Yin, H., 2009. Experimental transmission of Theileria annulata surface protein (TaSP) closely related to PIM of Theileria
Theileria uilenbergi infective for small ruminants by Haemaphysalis longicornis and parva: implications for use in diagnostic tests and subunit vaccines. Mol.
Haemaphysalis qinghaiensis. Parasitol. Res. 104, 1227–1231. Biochem. Parasitol. 120, 247–256.
Liu, Z., Dang, Z., Luo, J., Yin, H., Ahmed, J.S., Seitzer, U., 2008a. Small-scale expressed Schnittger, L., Yin, H., Gubbels, M.J., Beyer, D., Niemann, S., Jongejan, F., Ahmed, J.S.,
sequence tag analysis of Theileria uilenbergi: identification of a gene family 2003. Phylogeny of sheep and goat Theileria and Babesia parasites. Parasitol. Res.
encoding potential antigenic proteins. Ann. NY Acad. Sci. 1149, 214–217. 91, 398–406.
Liu, Z., Hou, J., Bakheit, M.A., Salih, D.A., Luo, J., Yin, H., Ahmed, J.S., Seitzer, U., 2008b. Schnittger, L., Yin, H., Qi, B., Gubbels, M.J., Beyer, D., Niemann, S., Jongejan, F.,
Development of loop-mediated isothermal amplification (LAMP) assay for rapid Ahmed, J.S., 2004. Simultaneous detection and differentiation of Theileria and
diagnosis of ovine theileriosis in China. Parasitol. Res. 103, 1407–1412. Babesia parasites infecting small ruminants by reverse line blotting. Parasitol.
Luo, J., Yin, H., 1997. Theileriosis of sheep and goats in China. Trop. Anim. Health Res. 92, 189–196.
Prod. 29, 8S–10S. Seitzer, U., Liu, Z., Yin, H., Beyer, D., Kullmann, B., Miranda, J., Ahmed, J.S., 2008.
Miranda, J., Bakheit, M.A., Liu, Z., Yin, H., Mu, Y., Guo, S., Beyer, D., Oliva, A., Ahmed, Immune response of Theileria sp.-infected sheep to recombinant Theileria
J.S., Seitzer, U., 2006a. Development of a recombinant indirect ELISA for the proteins. Ann. NY Acad. Sci. 1149, 186–190.
diagnosis of Theileria sp. (China) infection in small ruminants. Parasitol. Res. 98, Yin, H., Liu, Z., Guan, G., Liu, A., Ma, M., Ren, Q., Luo, J., 2008. Detection and
561–567. differentiation of Theileria luwenshuni and T. uilenbergi infection in small
Miranda, J.P., Bakheit, M., Schneider, I., Haller, D., Ahmed, J.S., Yin, H., Oliva, A.G., ruminants by PCR. Transbound. Emerg. Dis. 55, 233–237.
Seitzer, U., 2006b. Identification of homologous genes of T. annulata proteins in Yin, H., Luo, J., Guan, G., Gao, Y., Lu, B., Zhang, Q., Ma, M., Lu, W., Lu, C., Yuan, Z., Guo,
the genome of Theileria sp. (China). Ann. NY Acad. Sci. 1081, 468–470. S., Wang, B., Du, H., Schnittger, L., Ahmed, J., Jongejan, F., 2002. Transmission of
Nagore, D., Garcia-Sanmartin, J., Garcia-Perez, A.L., Juste, R.A., Hurtado, A., 2004. an unidentified Theileria species to small ruminants by Haemaphysalis
Identification, genetic diversity and prevalence of Theileria and Babesia species qinghaiensis ticks collected in the field. Parasitol. Res. 88, S25–S27.
in a sheep population from Northern Spain. Int. J. Parasitol. 34, 1059–1067. Yin, H., Liu, G., Luo, J., Guan, G., Ma, M., Ahmed, J., Bai, Q., 2003. Observation on the
Pain, A., Renauld, H., Berriman, M., Murphy, L., Yeats, C.A., Weir, W., Kerhornou, A., schizont stage of an unidentified Theileria sp. in experimentally infected sheep.
Aslett, M., Bishop, R., Bouchier, C., Cochet, M., Coulson, R.M., Cronin, A., de Parasitol. Res. 91, 34–39.
Villiers, E.P., Fraser, A., Fosker, N., Gardner, M., Goble, A., Griffiths-Jones, S., Yin, H., Luo, J., Schnittger, L., Lu, B., Beyer, D., Ma, M., Guan, G., Bai, Q., Lu, C., Ahmed,
Harris, D.E., Katzer, F., Larke, N., Lord, A., Maser, P., McKellar, S., Mooney, P., J., 2004. Phylogenetic analysis of Theileria species transmitted by Haemaphysalis
Morton, F., Nene, V., O’Neil, S., Price, C., Quail, M.A., Rabbinowitsch, E., Rawlings, qinghaiensis. Parasitol. Res. 92, 36–42.
N.D., Rutter, S., Saunders, D., Seeger, K., Shah, T., Squares, R., Squares, S., Tivey, Yin, H., Schnittger, L., Luo, J., Seitzer, U., Ahmed, J.S., 2007. Ovine theileriosis in
A., Walker, A.R., Woodward, J., Dobbelaere, D.A., Langsley, G., Rajandream, M.A., China: a new look at an old story. Parasitol. Res. 101 (Suppl. 2), S191–S195.