Molecular detection of Theileria and Babesia infections in cattle§
Kursat Altay, M. Fatih Aydin, Nazir Dumanli, Munir Aktas*
Department of Parasitology, Faculty of Veterinary Medicine, University of Firat, 23119 Elazig, Turkey
Received 31 May 2008; received in revised form 27 August 2008; accepted 15 September 2008
This study was carried out to determine the presence and distribution of tick-borne haemoprotozoan parasites (Theileria and
Babesia) in apparently healthy cattle in the East Black Sea Region of Turkey. A total of 389 blood samples were collected from the
animals of various ages in six provinces in the region. Prevalence ofinfection was determined by reverse line blot (RLB) assay. The
hypervariable V4 region of the 18S ribosomal RNA (rRNA) gene was amplified with a set of primers for members of the genera
Theileria and Babesia. Amplified PCR products were hybridized onto a membrane to which generic- and species-specific
also examined microscopically for Theileria and/or Babesia spp. and 5.14% were positive. All samples shown to be positive by
microscopy also tested positive with RLB assay. Two Theileria (T. annulata and T. buffeli/orientalis) and three Babesia (B.
bigemina, B. major and Babesia sp.) species or genotypes were identified in the region.Babesia sp. genotype shared 99% similarity
with the previously reported sequences of Babesia sp. Kashi 1, Babesia sp. Kashi 2 and Babesia sp. Kayseri 1. The most frequently
found species was T. buffeli/orientalis, present in 11.56% of the samples. T. annulata was identified in five samples (1.28%).
and Babesia sp. in five samples (1.28%). A single animal infected with T. buffeli/orientalis was also infected with B. bigemina.
# 2008 Elsevier B.V. All rights reserved.
Keywords: Theileria; Babesia; PCR; Reverse line blot; Cattle; Turkey
Theileria and Babesia species are tick-borne
haemoprotozoan parasites of vertebrates that have a
major impact on livestock production, mainly cattle and
small ruminants, in tropical and subtropical areas
(Mehlhorn and Schein, 1984). Theileria annulata and
high morbidity and mortality, whereas Theileria buffeli/
orientalis causes mild or asymptomatic disease in
cattle. Bovine babesiosis is caused by Babesia
bigemina, Babesia bovis, Babesia divergens and
Babesia major. Babesia species have the potential for
wide distribution wherever their tick vectors are
encountered. Two species, B. bovis and B. bigemina,
have a considerable impact on cattle health and
productivity in tropical and subtropical countries
Techniques for detection of these haemoparasites
have been developed separately for use in each species.
The traditional method of identifying the agents in
smears stained with Giemsa. This technique is usually
adequate for detection of acute infections, but not for
Available online at www.sciencedirect.com
Veterinary Parasitology 158 (2008) 295–301
§Nucleotide sequence data reported in this paper are available in
GenBank, EMBL and DDBJ databases under accession numbers from
EU622821 to EU622825.
* Corresponding author. Tel.: +90 424 237 0000;
fax: +90 424 238 8173.
E-mail address: firstname.lastname@example.org (M. Aktas).
0304-4017/$ – see front matter # 2008 Elsevier B.V. All rights reserved.
employed in diagnosing subclinical infections in
epidemiological studies, but false-positive and false-
negative results, due to cross-reactions or weakening of
specific immune responses, are common (Passos et al.,
1998). Therefore, a sensitive and highly specific method
for the diagnosis of piroplasms is required. Recently,
species-specific polymerase chain reaction (PCR) and
PCR-based reverse line blot (RLB) hybridization
methods have been developed for the detection and
et al., 1992; Calder et al., 1996; Gubbels et al., 1999;
Georges et al., 2001; Aktas et al., 2005; Garcı ´a-
Sanmartı ´n et al., 2006; Altay et al., 2007a; M’ghirbi
et al., 2008).
The main tick-borne haemoparasitic diseases occur-
ring in cattle throughout Turkey are theileriosis and
babesiosis. Bovine theileriosis has been investigated
using molecular techniques, and the presence of T.
annulata and T. buffeli/orientalis has been reported in
the some part of the country (Aktas et al., 2002, 2006;
Dumanli et al., 2005). B. bovis, B. bigemina and B.
divergens have been detected by microscopy and
serological tests (Aktas et al., 2001). However, these
methods are less sensitive and specific in the detection
of carrier animals and do not generally distinguish
between current infections and previous exposures.
Identification of carrier animals is important for the
assessment of infection risk. They serve as reservoirs of
infection for ticks and cause natural transmission of the
disease (Calder et al., 1996). PCR-based techniques
provide an alternative method for the direct detection of
piroplasms in carrier animals. In the present study, a
molecular survey of Theileria and Babesia species,
based on PCR amplification and RLB hybridization,
was conducted in cattle in the East Black Sea Region of
Turkey. The results of RLB were compared to those of
examination of thin blood smears.
2. Materials and methods
2.1. Study area and collection of samples
The study was conducted on cattle in the provinces
of Tokat, Amasya, Gumushane, Giresun, Trabzon and
Rize located in the East Black Sea Region of Turkey
(Fig. 1). This area has varying weather conditions.
Unlike the dry and hot East and Central Anatolia
regions, the East Black Sea region covers two different
climatic zones: an Atlantic climate in the coastal region
with frequent rainfall and mild temperatures. The main
tick species here are Ixodes ricinus, Haemaphysalis
punctata, Haemaphysalis sulcata, Dermacentor mar-
ginatus, Rhipicephalus bursa and a Continental
Mediterranean climate in the interior regions, with
warmer summers and colder winters. The main tick
species here are Hyalomma marginatum marginatum,
Boophilus annulatus, Rhipicephalus spp.
Blood samples from 389 cattle randomly selected
from 74 farms, also randomly selected, were taken in
EDTA containing tubes. A thin blood smear from each
sample was prepared and numbered in the field by the
same person. The blood samples were stored at ?20 8C
until DNA extraction. The age of animals ranged
between 1 and 7 years, and all were clinically healthy.
2.2. Microscopic examination
In the laboratory, the blood smears were fixed in
methanol for 5 min and stained for 30 min in Giemsa
K. Altay et al./Veterinary Parasitology 158 (2008) 295–301 296
Fig. 1. Map of the Turkish provinces, showing the locations surveyed in the current study.
stain diluted with 5% buffer. Slides were examined for
intra-erythrocytic forms of Theileria and Babesia spp.
piroplasms at 100? objective magnification. Approxi-
mately 20 000 erythrocytes per slide were examined for
the calculation of percentage of infected erythrocytes.
The smears were recorded as negative for piroplasms if
no parasites were detected in 50 oil-immersion fields.
2.3. DNA extraction
DNA extraction was performed as described by
d’Oliveira et al. (1995). Briefly, 125 ml of blood was
addedto250 mloflysissolution(0.32 Msucrose,0.01 M
Tris, 0.005 M MgCl2, 1% Triton X-100, pH 7.5). The
mixture was centrifuged at 11 600 ? g for 1 min. The
pellet was washed three times by centrifugation with
the final pellets were resuspended in 100 ml of PCR
buffer (50 mM KCl, 10 mM Tris–HCl (pH 8), 0.1%
Triton X-100, pH 8.3). Proteinase K (50 mg/ml) was
added to the pellet suspension, and the mixture was
incubated at 56 8C for 1 h. Finally, the samples were
heated at 100 8C for 10 min.
2.4. 18S rRNA gene amplification and RLB
For the amplification of Theileria and Babesia
species, one set of primers was used to amplify an
approximately 390–430 bp fragment of the hypervari-
able V4 region of the 18S rRNA gene. The forward
and the reverse [RLB-R2 (Biotin-50-CTAAGAATTT-
CACCTCTGACAGT-30)] primers were as described by
Georges et al. (2001). The PCR volume and reaction
conditions applied were similar to those described by
Altay et al. (2007a). The primers and oligonucleotide
probes (catchall Theileria/Babesia, Theileria spp., T.
buffeli/orientalis, T. annulata, Babesia spp., B. bige-
mina, B. bovis, B. divergens, B. major), containing an
pyl phosphoramidite (TFA)-C6 aminolinker were
synthesised by Isogen, Maarssen, Netherlands. Pre-
paration, hybridization and stripping of RLB membrane
were performed as described by Altay et al. (2007a).
2.5. Sequencing and phylogenetic analysis
To confirm RLB results, representative PCR pro-
ducts were chosen randomly for sequencing. Generated
DNA fragment of approximately 390 and 430 bp of
Theileria and Babesia were extracted from 1.5%
agarose gel using a commercial kit (Wizard SV gel
and PCR clean-up system, Promega, Madison, WI,
USA). The purified PCR products were sequenced and
submitted to GenBank. Each construct was sequenced
at least three times and subjected to BLAST similarity
searches. A phylogenetic tree was created from the
sequences of the 18S rRNA genes of cattle Babesia
species identified in this study and those available from
GenBank, using the neighbour-joining method in
MEGAversion 3.1 (Kumar et al., 2004). The nucleotide
sequences used in this study are available in GenBank
under the following accession numbers: AY726556 for
Babesia sp. Kashi 1; EF434786 for Babesia sp. Kayseri
1; AY726557 for Babesia sp. Kashi 2; AY596729 for B.
orientalis; AY603400, AY603401 and AY081192 for B.
ovata; DQ785311 and EF612434 for B. bigemina;
AY648886 and AY603339 for B. major; AY789076 and
AY572456 for B. divergens; AF316893 for Plasmodium
2.6. Statistical analysis
A x2-squared test was used to evaluate the
differences among various parameters. P < 0.05 was
accepted to be statistically significant.
3.1. Specificity of the RLB assay
Primers RLB-F2 and RLB-R2 amplified bands of
?390 and ?430 bp corresponding to the hypervariable
V4 region of the 18S rRNA gene of Theileria and
Babesia species. PCR performed on uninfected control
cattle DNA and a water control did not yield detectable
product on agarose gel (data not shown). All PCR
positive samples showed positive reactions with their
corresponding specific probes. However, some samples
gave positive signals to catchall and Babesia genera-
specific probes, but did not show any signal to the
species-specific probes tested (Fig. 2). This situation
indicated the presence of a novel Babesia genotype.
3.2. Sequencing and phylogenetic analysis
Two Theileria and three Babesia sequences were
identified. The partial sequences of the 18SrRNA genes
for T. buffeli/orientalis, B. bigemina, T. annulata,
Babesia sp. CS58 and B. major were deposited in the
EMBL/GenBank databases under accession numbers
from EU622821 to EU622825, respectively. Theileria
sequences shared 99% identity with the recently
K. Altay et al./Veterinary Parasitology 158 (2008) 295–301 297
reported sequences for the 18S rRNA gene of T. buffeli/
orientalis (EU407247) and T. annulata (AY508473).
From the three Babesia sequences, two of which were
most closely related to the B. bigemina and B. major,
and they shared 99% identity to recently reported
sequences for B. bigemina (EF612434) and B. major
(AY603339). The third sequence differed clearly from
the all known Babesia species infective for cattle, but
shared 99% similarity with the unnamed Babesia
isolates (Babesia sp. Kashi 1, Babesia sp. Kashi 2 and
Babesia sp. Kayseri 1) and B. orientalis.
The constructed phylogenetic tree revealed that
Babesia species infective to cattle split into five
monophyletic clades. Babesia sp. CS58 identified in
this study demonstrated a close relationship and was
included in the clade with unnamed Babesia isolates
(Babesia sp. Kashi 1, Babesia sp. Kashi 2 and Babesia
sp. Kayseri 1) and B. orientalis (Fig. 3).
3.3. Prevalence of piroplasm infections in cattle
Thin blood smears revealed parasitaemia in infected
cattle ranging from 0.01% to 0.1%. Piroplasms,
detected inside erythrocytes, were pleomorph and ring-
or pear-shaped. Prevalence of piroplasms detected by
microscopy and RLB from samples at locations in the
the 389 blood samples examined, microscopy revealed
20 (5.14%) positive for piroplasms, whereas 63
(16.19%) of DNA amplified products hybridized with
the probes for catchall, and genera- and species-specific
probes. These results demonstrated that RLB showed a
significantly higher rate of detection of Theileria and
Babesia infections (P < 0.01) than did microscopic
examination. In the RLB and microscopy analysis, the
highest number of positive samples and the highest
carriers of piroplasms were obtained from the province
of Tokat with 31.08% and 9.45%.
Prevalence of each piroplasm species identified in
cattle is shown in Table 2. Theileria spp. prevalencewas
12.85% (50/389), and prevalence of Babesia spp. was
2.57% (10/389). The most frequently found species was
annulata was identified in five samples (1.28%).
Babesia infections were less frequently detected:
Babesia sp. were found in five samples (1.28%); B.
bigemina in three samples (0.77%) and B. major in two
samples (0.51%), one animal infected with T. buffeli/
orientalis was also infected with B. bigemina (Fig. 2,
Cattle with subclinical theileriosis and babesiosis
become chronic carriers of the piroplasm and, hence,
sources of infection for tick vectors. Therefore, latent
infections are important in the epidemiology of the
diseases. The diagnoses of piroplasm infections are
based on clinical findings and microscopic examination
of Giemsa-stained blood smears. However, this method
is not sensitive enough or sufficiently specific to detect
chronic carriers, particularly when mixed infections
occur. Molecular techniques enable sensitive and
specific detection of the parasites. The RLB method
is an effectiveand practical tool, since it isable to detect
extremely low parasitaemia levels and simultaneously
identify Theileria and Babesia species using specific
oligonucleotide probes (Gubbels et al., 1999; Altay
K. Altay et al./Veterinary Parasitology 158 (2008) 295–301298
Fig. 2. Reverse line blot assay of the PCR products generated by amplification of genomic DNA from cattle samples infected with Theileria and
Samples bearing identified single and mixed infections and negative and positive controls are showed as follows: lane 1, T. buffeli/orientalis; lane 2,
T. annulata; lane 3, B. bigemina (field sample); lane 4, B. bovis (positive template); lane 5, B. divergens (positive template); lane 6, B. major (field
sample); lane 7, Babesia sp. (field sample); lane 8, T. buffeli/orientalis and B. bigemina (field sample); lane 9, negative control (genomic DNA of
uninfected cattle); lane 10, negative PCR control; lane 11, T. buffeli/orientalis 18S rRNA gene sequence; lane 12, T. annulata 18S rRNA gene
sequence; lane 13, B. bigemina 18S rRNA gene sequence; lane 14, B. major 18S rRNA gene sequence; lane 15, Babesia sp. CS58 18S rRNA gene
et al., 2007a). In this study, a molecular survey based on
PCR amplification and RLB hybridization was per-
formed for detection of bovine Theileria and Babesia
species. By this method, 63 of 389 (16.19%) samples
examined showed a positive signal to one or more
species-specific probes as well as to the corresponding
genus-specific probes. It was reported that the
oligonucleotide probes used in this study reacted with
the exception of the T. lestoquardi-specific probe which
cross-reacted with T. annulata (Nagore et al., 2004;
Altay et al., 2007a). T. lestoquardi infects sheep and
goats and has not been reported in Turkey (Altay et al.,
The survey identified different two Theileria (T.
annulata, T. buffeli/orientalis) and three Babesia (B.
bigemina, B. major, and a new Babesia genotype)
species and genotypes infecting cattle. The survey
revealed that the most frequently found species was T.
buffeli/orientalis, present in 11.56% of the samples. We
also foundthat T.annulatawas present inthe same area,
but the prevalence of this species was lower (1.28%).
These results are not in agreement with previous studies
carried out in Eastern Turkey (Dumanli et al., 2005;
Aktas et al., 2006). The lower prevalence of T. annulata
compared to T. buffeli/orientalis was related to the
geographic distribution of the tick vectors associated
K. Altay et al./Veterinary Parasitology 158 (2008) 295–301299
Light-microscopy examination of thin blood smears and RLB results
(Bovine Theileria and Babesia) by locations in the East Black Sea
Region of Turkey.
Total38920 5.1463 16.19
n, number of samples; +, positive samples.
Fig. 3. Neighbour-joining analysis of the 18S rRNA gene of the bovine Theileria and Babesia identified in this study and those present in the
GenBank database. Numbers above the branch demonstrate bootstrap support from 1000 replications. The tree was created using the MEGA 3.1
Scale bar represents nucleotide substitutions per position.
with each species. In the present study, Hyalomma
anatolicum anatolicum, the main vector tick of T.
annulata in Turkey, was not found on cattle, whereas
Haemaphysalis spp. were the dominant tick species
The prevalence of Babesia infection was lower than
that of Theileria infection. In a previous serological
study in the same region, serum antibodies against B.
bigemina, B. bovis and B. divergens were detected in
et al., 1991). In the present study, the lower prevalence
of Babesia species detected among carrier cattle as
compared to carriers of Theileria species could be
explained by the fluctuations in parasitaemia that occur
in the chronic phase of infection by Babesia species
(Calder etal., 1996; Gubbels et al., 1999). This situation
could also be explained by the low number of intra-
erythrocytic piroplasms circulating in the bloodstream
of Babesia carriers (Homer et al., 2000).
By sequencing the 18S rRNA gene of the Babesia
isolates identified in this study, a phylogenetic tree was
created (Fig. 2). It showed that the Babesia sp. CS58
isolate was in the clade with the unidentified Babesia
isolates from China (Kashi 1 and Kashi 2) and Turkey
(Kayseri 1) as well as with B. orientalis. Sequence
comparisons (357 nucleotides) of Babesia sp. CS58
revealed that the isolate differed clearly from all known
Babesia species infective for cattle but shared 99%
similarity with the Babesia sp. Kashi 1 and Kashi 2
isolated from H. anatolicum anatolicum (Luo et al.,
2005) and with Babesia sp. Kayseri 1, isolated from H.
demonstrated that B. bigemina, B. major, and unnamed
Babesia sp. were present in cattle in Turkey. The
presence of B. bigemina was expected, since the
parasite has been reported previously (Dincer et al.,
1991). However, B. major was detected for the first time
in Turkey and therefore contributed greater insight into
bovine piroplasm distribution and phylogenetic diver-
sity. B. bovis and B. divergens were not detected in the
cattleexamined, although I.ricinus,B. annulatus andR.
bursa, the vector ticks of these species, were identified
among the tick collected (unpublished data). The
absence of these species in RLB can possibly be
explained by fluctuations in low level parasitaemia.
In conclusion, this study has revealed two Theileria
(T. annulata and T. buffeli/orientalis) and three Babesia
(B. bigemina,B. major andBabesia sp.)infecting cattle.
The RLB performed has revealed a novel bovine
Babesia genotype. The assay provided more accurate
data on prevalence of infection and allowed direct
identification of species.
This work was supported financially by a grant (106
O 416) from the Scientific and Technical Research
Council of Turkey (TUBITAK).
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