Veterinary Parasitology 133 (2005) 277281

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Development of a polymerase chain reaction method for

diagnosis of Babesia ovis infection in sheep and goats
M. Aktas *, K. Altay, N. Dumanl
Department of Parasitology, Faculty of Veterinary Medicine, University of Firat, 23119 Elazig, Turkey
Received 3 May 2005; received in revised form 6 May 2005; accepted 27 May 2005

Abstract
In this study, a pair of oligonucleotide primers were designed according to the nucleotide sequence of the small subunit
ribosomal RNA (ssu rRNA) gene of Babesia ovis isolated from sheep in eastern Turkey. The primers were used to detect parasite
DNA from blood samples of B. ovis-infected sheep and goats by polymerase chain reaction (PCR). A 549-bp DNA fragment was
specifically amplified from blood samples from sheep and goats, naturally infected with B. ovis. No PCR products resulted from
Babesia motasi, T. ovis, Theileria sp. OT1, Theileria sp. OT3, T. lestoquardi, B. canis, B. microti, T. annulata or normal sheep
leucocytes DNA using these specific primers. B. ovis-infected erythrocytes with 1% parasitemia were subjected to 10-fold serial
dilutions (from 10
1 to 10
9) using an uninfected sheep erythrocytes, and DNA was extracted from each diluted sample for
testing the sensitivity of the PCR. The PCR was sensitive enough to detect parasite DNA from the dilution of 10
5 with
0.00001% parasitemia. This is more sensitive than examining 200 fields under light microscopy. In addition, 98 field samples
collected from small ruminanats in eastern Turkey were tested for B. ovis infection. Four samples were positive Babesia spp. in
blood smears, 21 samples were positive for B. ovis DNA by PCR. These results indicate that the PCR provides a useful diagnostic
tool for the detection of B. ovis infection in sheep and goats.
# 2005 Elsevier B.V. All rights reserved.
Keywords: Babesia ovis; Sheep and Goats; PCR; Microscopy; Specific primers

1. Introduction
Ovine babesiosis is the most important hemoparasitic tick-borne disease of small ruminants caused
by Babesia ovis, Babesia motasi and Babesia crassa.
These parasites are widespread in tropical and
subtropical areas of the world (Uilenberg, 2001).
* Corresponding author. Tel.: +90 424 237 0000;
fax: +90 424 238 8173.
E-mail address: maktas@firat.edu.tr (M. Aktas).

B. ovis is highly pathogenic especially in sheep and

causes severe infections which is charecterised by
fever, anemia, icterus and hemoglobinuria. Mortality
rates in susceptible hosts range from 30 to 50% in field
infections. The pathogenecity of B. motasi is not high
and appears to be moderately virulent. In contrast, B.
crassa is considered as being non-pathogenic to small
ruminants (Friedhoff, 1997; Hashemi-Fesharki, 1997).
Microscopic examination of Giemsa stained blood
smears remains the most appropriate for the diagnosis
of acute babesiosis, but the low sensitivity of the

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M. Aktas et al. / Veterinary Parasitology 133 (2005) 277281

method does not permit its use in epidemiological

investigations (Almeria et al., 2001). Serological
methods are frequently employed in determining
subclinical infections in epidemiological studies.
However, these methods lack specificity due to cross
reactivity with other Babesia species (Passos et al.,
1998). Furthermore, false positive and negative results
are commonly observed in these tests. Therefore,
development of a highly specific and sensitive method
for the diagnosis of Babesia infections is required.
Recently, molecular techniques have become the
preferred methods for diagnosis of babesiosis and
theileriosis, because these techniques are more
sensitive and specific than other conventional methods
(Nagore et al., 2004; Schnittger et al., 2004; Alhassan
et al., 2005; Altay et al., 2005).
In this study, the use of PCR for the specific
detection of B. ovis in sheep and goats infected
erythrocytes using oligonucleotides designed from the
sequence of the small subunit ribosomal RNA (ssu
rRNA) gene of B. ovis isolated from sheep in eastern
Turkey is described.

2. Materials and methods

Blood samples were obtained from two sheep with
clinical babesiosis in Elazig province, Turkey. DNAs
extracted from the samples were revealed to be
Babesia ovis by ssu rRNA gene sequence analysis.
The nucleotide sequences of ssu rRNA gene of B. ovis
have been submitted to the GenBank data base under
accession no. AY998123 and AY998124. In this study,
DNAs from these samples were used as positive
controls for B. ovis specific PCR. Plasmid DNAs of
Babesia motasi, Theileria sp. OT1 and Theileria sp.
OT3 were kindly provided by Dr. Ana Hurtado
(Department of Animal Health, Instituto Vasco de
Investigacion y Desarrollo Agrario (NEIKER) Berreaga 1, 48160 Derio, Bizkaia, Spain). Genomic DNA
of T. lestoquardi was kindly provided by Prof. Dr. J.S.
Ahmed (Department of Immunology and Cell biology,
Research Center, Borstel, Germany) and Babesia
canis and Babesia microti were kindly provided by Dr.
Bernard Carcy (Faculte de Pharmacie, Laboratoire de
Biologie Cellulaire et Moleculaire, Universite Montpellier, France). T. annulata and T. ovis were isolated
from a cow and sheep at our laboratory (Department of

Parasitology, Faculty of Veterinary Medicine, University of Firat, Elazig, Turkey). Venous blood sample,
taken from a healthy sheep without contact with ticks,
was used as the negative control.
Extraction of DNA was performed according to the
method described by Clausen et al. (1999) with minor
modifications. Briefly, 125 ml of blood was added to
250 ml of lysis mixture (0.32 M sucrose, 0.01 M Tris,
0.005 M MgCl2, 1% Triton X-100, pH 7.5) and the
mixture was centrifuged at 11,600  g for 1 min. The
pellet was washed three times by centrifugation with
250 ml lysis buffer. The supernatants were discarded
and the final pellets were resuspended in 100 ml of
PCR buffer (50 mM KCl, 10 mM TrisHCl (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 then incubated at 56 8C for 1 h. Lastly, the sample
was boiled at 95 8C for 10 min.
Two oligonucleotide primer targets for the 549-bp
fragment were designed based on the ssu rRNA gene
sequence of B. ovis isolated from sheep in eastern
Turkey (GenBank accession numbers AY998123 and
AY998124). Forward strand primer Bbo-F 50 TGGGCAGGACCTTGGTTCTTCT-30 , and reverse
strand primer Bbo-R 50 -CCGCGTAGCGCCGGCTAAATA-30 were used in the PCR amplification.
The PCR was performed in a touchdown thermocycler in a total reaction volume of 50 ml containing
5 ml of 10X PCR buffer [750 mM TrisHCl (pH 8.8),
200 mM (NH4)2SO4, 0.1% Tween 20], 2 mM MgCl2,
250 mM of each of the four deoxynucleotide triphosphates, 1.25 U Taq DNA polymerase (Fermentas) and
50 pmol of each primer and 5 ml of template DNA.
The reaction mixture was overlaid with 100 ml
mineral oil and amplification was carried out in a
no hot-lid minicycler (MJ Research, US). The reaction
was repeated for 35 cycles under the following
conditions: 1 min at 94 8C, 1 min at 62 8C and 1 min at
72 8C. PCR products were visualized by UV
transillumination in a 1.5% agarose gel following
electrophoresis and staining with ethidium bromide.
DNAs from Babesia motasi, T. ovis, Theileria sp.
OT1, Theileria sp. OT3, T. lestoquardi, B. canis, B.
microti, T. annulata, and blood from a normal sheep
with no exposure to ticks were tested in the PCR along
with B. ovis DNA using the procedure described
above. Primers RLB-F and -R (50 -GAGGTAGTGACAAGAAATAACAATA-30 and 50 -TCTTCGATCCC-

M. Aktas et al. / Veterinary Parasitology 133 (2005) 277281

CTAACTTTC-30 , described as primers for specific

amplification (460520-bp) of Babesia and Theileria
species 18S ssu rRNA genes (Gubbels et al., 1999),
were used in the PCR assay as a positive quality
control for the Babesia and Theileria species.
To determine the detection limit of the PCR assay,
B. ovis-infected erythrocytes with 1% parasitemia
calculated according to Oura et al. (2004) were
subjected to 10-fold serial dilutions (from 10
1 to
10
9) using an uninfected sheep erythrocytes, and
DNA was extracted from each diluted sample and
processed for PCR as described above.
A field study was conducted in Elazig province of
eastern Turkey, to assess the suitability of the PCR for
use with field samples. Blood samples were collected
in tubes with EDTA from 98 small ruminants (68
sheep, 30 goats). These samples were used to prepare
blood smears for microscopic examination and to
extract DNA for PCR analysis. The blood smears were
prepared immediately after taking the blood samples.
The blood smears were fixed with methanol for 5 min,
stained with Giemsa at a dilution of 5% in buffer
solution for 30 min, and then examined at 1000
magnification for the presence of Babesia piroplasms
by microscopy. The blood smears were recorded as
negative for Babesia spp., if no piroplasms were
observed in 200 oil-immersion fields.

3. Results
The specificity of the primer pair was tested with
DNA from B. ovis, B. motasi, B. canis, B. microti,
Theileria sp. OT1, Theileria sp. OT3, T. lestoquardi, T.
ovis and T. annulata. The expected 549-bp fragment
was generated from B. ovis DNA, but was not
generated from the other Babesia and Theileria DNAs
(Fig. 1B). The Babesia and Theileria-specific primers
RLB-F/-R were used as positive control and amplified
a 460520-bp fragment from the Babesia and
Theileria species examined (Fig. 1A). The results of
the detection limit of the assay are shown in Fig. 2. The
blood dilutions between 1 and 10
5% are clearly
positive with a diminishing signal as the target DNA
concentration decreases. The lowest concentration of
piroplasm detected was 0.00001% parasitemia in
PCR. The PCR results for B. ovis showed that one
infected cell out of 107 sheep erythrocytes or that

279

Fig. 1. Specificity of the PCR method. Ethidium bromide stained

agarose gel electrophoresis of amplication products from Babesia
and Theileria species using Babesia and Theileria-specific primers
or B. ovis-specific primers. (A) PCR with Babesia- and Theileriaspecific primer pair RLB-F/-R. (B) PCR with B. ovis-specific primer
pair Bbo-F and -R. Lane M, 100-bp ladder DNA marker; lane 1,
uninfected sheep blood (negative control); lane 2, B. ovis; lane 3, B.
motasi; lane 4, T. ovis; lane 5, Theileria sp. OT1; lane 6, Theileria sp.
OT3; lane 7, T. lestoquardi; lane 8, B. canis; lane 9, B. microti; lane
10, T. annulata.

parasites in blood with an equivalent parasitemia of

0.00001% could be detected.
Thin blood smear examination of 98 small
ruminants (68 sheep, 30 goats) from eastern Turkey
showed that four animals (three sheep, one goat) were
positive for Babesia spp. piroplasms. Using PCR, 21
of 98 (21.42%) animals (19 sheep, two goats) were
positive. All of the positive samples by thin blood
smears were also determined to be positive by PCR,
whereas no piroplasms were seen by light microscopy
in 17 of the 21 PCR positive animals.

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M. Aktas et al. / Veterinary Parasitology 133 (2005) 277281

Fig. 2. Sensitivity of the PCR method. Ethidium bromide stained

agarose gel electrophoresis of amplication products from 10-fold
serial diluted samples (from 10
1 to 10
9%). Lane M, 100-bp ladder
DNA marker; lane 1, undiluted blood with 1% parasitemia; lane 2,
dilution of 10
1 with 0.1% parasitemia; lane 3, dilution of 10
2 with
0.01% parasitemia; lane 4, dilution of 10
3 with 0.001% parasitemia; lane 5, dilution of 10
4 with 0.0001% parasitemia; lane 6,
dilution of 10
5 with 0.00001% parasitemia; lane 7, dilution of 10
6
with 0.000001% parasitemia; lane 8, dilution of 10
7 with
0.0000001% parasitemia; lane 9, dilution of 10
8 with
0.00000001% parasitemia; lane 10, dilution of 10
9 with
0.000000001% parasitemia; lane 11, positive control; lane 12,
uninfected sheep blood (negative control).

4. Discussion
The diagnosis of piroplasm infections in vertebrate
hosts has been mainly carried out by microscopic
examination of blood smear. However, the method
requires expertise because these parasites have similar
morphological features and therefore may confuse
when mixed infections occur. Serological tests have
also been used, but there are some difficulties with
specificity and sensitivity (Passos et al., 1998). An
exact differentiation between these parasites is crucial
to understand their epidemiology.
The detection of Babesia infection in carrier animals
by DNA amplification was a powerful tool for
epidemiological investigation, since these animals
represent an important source of alimentary infection
of Ixodid ticks. It is more sensitive and specific than
detection of parasite by conventional methods (dOliveira et al., 1995; Aktas et al., 2002). Amplification of
parasite DNA using specific PCR has been applied to
various Babesia and Theileria species (Kirvar et al.,
1998, 2000; Schnittger et al., 2000; Fukumoto et al.,
2001; Oliveira-Sequira et al., 2005; Alhassan et al.,
2005; Altay et al., 2005), but PCR specific for B. ovis

has not been available until now. In this study, a highly

specific and sensitive PCR assay was developed for the
detection of B. ovis in sheep and goats using a pair of
oligonucleotide primers which target the 549-bp
fragment in the ssu rRNA gene of the parasite.
The specificity of the primers used in this study for
B. ovis was determined by successful amplification of
a 549-bp product from positive control and collected
field samples. In contrast, no PCR products were seen
with B. motasi, B. canis, B. microti, Theileria sp. OT1,
Theileria sp. OT3, T. lestoquardi, T. ovis and T.
annulata DNAs. The successful amplification of only
B. ovis products indicates that the designed primers are
specific for B. ovis DNA. The Babesia and Theileriaspecific primers RLB-F/-R, described by Gubbels
et al. (1999), generated the expected a 460520-bp
fragment of Babesia and Theileria species examined,
including B. ovis, confirming the integrity of the
DNAs used to validate the specificity of the assay.
We found that the detection limit of the test was
0.00001% parasitemia. This is equal to a parasitemia
at the level of 10
5% and is over 100 times greater
than can be attained by microscopic examination of
blood smears. These results demonsrate that PCR
using B. ovis-specific primers is capable of detecting
B. ovis parasites present at extremely low parasitemias
in sheep and goats and can be applied to samples
collected under field conditions. Similar sensitivities
in Theileria and Babesia PCR assays have been
reported by dOliveira et al. (1995) for T. annulata,
Kirvar et al. (1998, 2000) for T. lestoquardi and T.
annulata, Fukumoto et al. (2001) for B. gibsoni,
Oliveira-Sequira et al. (2005) for B. bigemina and B.
bovis, Alhassan et al. (2005) for Babesia caballi and
Babesia equi, Altay et al. (2005) for T. ovis.
The results obtained from field samples indicated
that the frequency of B. ovis detected by DNA
amplification method was significantly higher than
detection by light microscopy, because the latter
method does not detect positive animals in the early
phase of infection and the long-term carrier status,
when the parasitemia is very low. The result agrees
with previous report about the B. bovis (Calder et al.,
1996). In conclusion, the primer pair described here
could be useful tools for clarifiying the epidemiology
of ovine babesiosis caused by B. ovis and for exact
discrimination between B ovis and B. motasi infections in sheep and goats.

M. Aktas et al. / Veterinary Parasitology 133 (2005) 277281

Acknowledgements
The authors would like to thank all veterinarians
and technicians for their kind help during sample
collection for this study. For the gifts of genomic DNA
from Jabbar Ahmed, Ana Hurtado and Bernard Carcy
are gratefully acknowledged. This study was supported by a grant (104 O 393) from The Scientific and
TAK).
BY
Technical Research Council of Turkey (TU

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