ANIMAL BIODIVERSITY AND EMERGING DISEASES

Comparison of the Molecular Structure of the

TaSP Gene of Theileria annulata from
Sudanese Isolates
Awadia M. Ali,a,b Diaeldin Salih,c Mohammed Bakheit,d
Abdel Rahim M. El Hussein,c Shawgi M. Hassan,a
Maowia M. Mukhtar,e Jabbar S. Ahmed,b and Ulrike Seitzerb
a
Faculty of Veterinary Medicine, University of Khartoum, Khartoum North, Sudan
b
Division of Veterinary Infection Biology and Immunology, Research Center Borstel,
Borstel, Germany
c
Central Veterinary Research Laboratories, Al Amarat, Khartoum, Sudan
d
National Research Center for Protozoan Diseases, Unit for Diseases Control and
Genetics, Obihiro University for Agriculture and Veterinary Medicine,
Inada-cho, Obihiro, Japan
e
Institute of Endemic Diseases, University of Khartoum, Khartoum, Sudan

The polymorphic region of the Theileria annulata surface protein (TaSP) was cloned
and sequenced from different isolates of cattle and cell lines from different areas of
Sudan. Amino acid sequence alignment revealed a high diversity showing amino acid
and length polymorphism, both within and between parasite isolates. The generation
of TaSP diversity may allow the evasion of host immunity by the parasite since TaSP is
a highly antigenic parasite protein.

Key words: alignment; genotype; Theileria annulata; Sudan

Introduction Materials and Methods

Theileria annulata, the causative agent of trop-
ical theileriosis, is a tick-borne protozoan para-
site. Tropical theileriosis constitutes one of the
obstacles for improved livestock production in
many countries. In the Sudan, the disease is
considered as the most important tick-borne
disease.1 No data is available regarding the
genotypes of this Theileria parasite in Sudan.
This study investigated the molecular struc-
ture of the single copy polymorphic T. annulata
surface protein (TaSP) gene2 for comparison
of strains isolated from cattle and cell lines in
Sudan.
Address for correspondence: Dr. Ulrike Seitzer, Veterinary Infection
Biology and Immunology, Research Center Borstel, Parkallee 22, 23845
Borstel, Germany. Voice: +49-(0)4537-188-413; fax +49-(0)4537-188-
627. useitzer@fz-borstel.de
Animal Biodiversity and Emerging Diseases: Ann. N.Y. Acad. Sci. 1149: 218–220 (2008).
doi: 10.1196/annals.1428.025  C 2008 New York Academy of Sciences.
Cell Lines and Field Samples
The parasite isolates and field samples
from different geographical regions of the
Sudan used in this study and the number of
clones analyzed per sample are summarized in
Table 1.
Sequence Analysis
Genomic DNA was amplified, cloned, and
sequenced as described previously.2 Analysis
of the variable region of TaSP was performed
by comparing the sequences from the various
isolates (GenBank accession EU032540-
EU032577). A total of 38 clones (one to four
clones of independent PCR reactions) were se-
quenced. The predicted amino acid sequences

218
Ali et al.: Molecular Structure of TaSP Gene of Theileria annulata 219

TABLE 1. Theileria annulata Isolates and Cell Lines from Different Geographical Locations in the Sudan

Isolate Origin Type n Clones analyzed

ATB Atbara (northern Sudan) Cell line, passage 75 1 4

HAN Hantoub (central Sudan) Cell line, passage 76 1 5
SHA Shambat (central Sudan) Cell line, passage 10 1 3
Field samples Khartoum (central Sudan) Field blood samples 14 26

TABLE 2. Summary of Alignment Results

Identical Strongly similar Weakly similar Different
Sample (alignment length) aa (%) aa (%) aa (%) aa (%)

All sequences, 17 isolates, 38 clones (141) 58 (41.13) 19 (13.48) 11 (7.8) 53 (37.59)

Field samples, 14 animals, 26 clones (143) 64 (44.76) 17 (11.89) 11 (7.69) 51 (35.66)
ATB cell line, 4 clones (134) 78 (58.21) 19 (14.18) 8 (5.97) 29 (21.64)
Field sample, one animal (1109), 4 clones (137) 90 (65.69) 19 (13.87) 6 (4.38) 22 (16.06)

were aligned using ClustalW available at PBIL
(Pôle Bio-Informatique Lyonnais) (http://
npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?
page=/NPSA/npsa_clustalw.html).3
Results
The sequence results showed size and se-
quence polymorphism for the TaSP polymor-
phic region. Nucleotide sequences were from
393 to 411 nucleotides, corresponding to de-
rived amino acid (aa) sequences of 131 to 137
residues. Alignment data retrieved from the
ClustalW analysis of all sequences obtained
showed an identity of 41.13% (58 aa residues),
a strong similarity of 13.48% (19 aa residues), a
weak similarity of 7.8% (11 aa residues), and
a difference of 37.59% (53 aa residues). There
was high diversity within and between parasite
isolates and variable sequences were detected
from one animal. No geographical specificity of
sequence types was observed. Alignment of the
field samples only gave an identity of 44.76%, a
strong similarity of 11.89%, a weak similarity of
7.69%, and a difference of 35.66%. Using se-
quence data derived from clones obtained from
one animal, the identity was 65.69%, strong
similarity 13.87%, weak similarity 4.38%, and
the difference 16.06%. Alignment of derived
amino acid sequences from a single cell line
(ATB) showed an identity of 78 (58.21%), a
strong similarity of 19 (14.18%), a weak simi-
larity of 8 (5.97%), and a difference in amino
acids of 29 (21.64%) over an alignment length
of 134 amino acids. Alignment results are sum-
marized in Table 2.
Discussion
The results demonstrated a variability of
TaSP in Sudanese T. annulata isolates. Interest-
ingly, variable sequences were obtained from
individual animals suggesting multiple clone
infection, which could be a result of the high
transmission rates that may allow genetic re-
combination of the clones. A similar sequence
diversity for a gene of T. annulata was previously
reported with respect to the major merozoite
antigen gene Tams 1 and also in T. parva for the
polymorphic immunodominant surface pro-
tein (PIM), which is closely related to TaSP.2,4,5
No significant association between TaSP se-
quences and their geographical origins were
found. The genetic data of the field isolates
220
indicate a panmictic population structure of
the parasite in the Sudan. This result was sup-
ported by the results obtained from the cell
lines that also showed different alleles in a
single culture at passage 75. This result could
explain the long duration needed to attenu-
ate some T. annulata strains isolated from en-
demic areas, which possibly consist of different
genotypes.
The genetic mechanisms generating the ob-
served polymorphism might be the intragenic
recombination taking place during the sexual
phase of the life cycle of the parasite in the
tick vector. Gene conversion, which has been
identified as a mechanism responsible for the
reshuffling of important epitopes to create novel
alleles, can not be excluded for the TaSP gene,
although the predominant location of T cell
epitopes is within the conserved regions of the
molecule.2,6 Gene conversion rather than in-
tragenic recombination was hypothesized to
be the main mechanism for the polymor-
phism observed in the T. parva surface protein
(PIM).4
The diversity in antigenic genes is usually the
result of positive selection pressure of the host
immune system, which may also be the case
for TaSP, which is a highly antigenic protein.7
In the TaSP molecule six peptide sequences are
located in the polymorphic region with binding
motifs to bovine major histocompatibility com-
plex (MHC) class I molecules.2 Therefore, the
generation of TaSP diversity could also allow
the evasion of host immunity. A previous study
using merozoite antigen (Tams 1) indicated the
selection of parasite genotypes during transmis-
sion by ticks, and that selection is mediated via
evasion of the host immune response.8 An in-
vestigation to confirm these possibilities in the
antigenic gene TaSP is needed in the future.
Annals of the New York Academy of Sciences
Acknowledgments
This research was partially supported by
DAAD (Deutscher Akademischer Austausch
Dienst), the European Union (ICA4-1999-
30151), Animal Resources Bank (Sudan), and
IAEA (International Atomic Energy Agency)
through technical project NO.SUD04/
027 entitled “Control of ticks and tick-borne
diseases in the Sudan.”
Conflicts of Interest
The authors declare no conflicts of interest.
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