This document summarizes a study that reports the first case of vertical transmission of Theileria lestoquardi in sheep. Researchers observed an outbreak of T. lestoquardi abortion and stillbirth in ewes, where approximately 35 late-term ewes lost their fetuses over 5 days. Samples from an aborted fetus and its ewe tested positive for T. lestoquardi via blood smears, tissue smears, and PCR analysis of liver and spleen samples. DNA sequencing of the PCR product showed 100% identity to the 18S rRNA gene of T. lestoquardi, confirming vertical transmission from the infected ewe to her fetus. This represents the first report of transplacental transmission of T.

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Veterinary Parasitology 203 (2014) 322–325
Contents lists available at ScienceDirect
Veterinary Parasitology
journal homepage: www.elsevier.com/locate/vetpar
Short Communication
Vertical transmission of Theileria lestoquardi in sheep
Amir Zakiana
, Mohammad Nouria,∗
, Farid Baratia
,
Hooman Kahrobab
, Abbas Jolodarc
, Fardokht Rashidid
a
Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahid Chamran University, Ahvaz, Iran
b
Department of Genetics, Faculty of Sciences, Shahid Chamran University, Ahvaz, Iran
c
Department of Basic Sciences, Faculty of Veterinary Medicine, Shahid Chamran University, Ahvaz, Iran
d
Graduated from Faculty of Veterinary Medicine, Shahid Chamran University, Ahvaz, Iran
a r t i c l e i n f o
Article history:
Received 27 September 2013
Received in revised form 18 March 2014
Accepted 5 April 2014
Keywords:
Theileria
Transplacental transmission
Ewe
Abortion
a b s t r a c t
This is the first report of an outbreak of Theileria lestoquardi abortion and stillbirth in a mob
of 450 ewes in July 2012, during which, approximately 35 late-term ewes lost their fetuses
over a 5-day period. A dead ewe and her aborted fetus were transported to the Ahvaz
Veterinary Hospital for the diagnostic evaluation. The microbial cultures from the ewe
vaginal discharges and fetal abomasal contents and the liver were negative. The blood films
of the ewe and her fetus contained Theileria piroplasms and the impression smears from ewe
liver and fetal spleen were positive for Theileria Koch blue bodies. The DNA was extracted
from the liver and spleen of ewe and her fetus, respectively, and analyzed by polymerase
chain reaction (PCR) using specific primers derived from the nucleotide sequences of 18S
ribosomal DNA (rDNA) gene of T. lestoquardi. A single fragment of 428-bp fragment was
amplified. The PCR product was directly sequenced and the alignment of the sequence
with similar sequences in GenBank®
showed 100% identities with 18S rDNA gene of T.
lestoquardi. The present study is the first report of the T. lestoquardi vertical transmission
that could be related to the abortion.
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
Generally, economic importance of malignant theilerio-
sis in sheep is related to mortality. The disease is active
in the tropical parts, the south and southwest, of Iran
(Hashemi-Fesharaki, 1997; Zaeemi et al., 2011). The ticks
of Hyalomma sp. are responsible for the transmission of the
disease in the region (Hooshmand Rad and Hawa, 1973).
Vertical transmission of theileriosis has been reported
in horse (Allsopp et al., 2007) and cows (Baek et al., 2003).
Intrauterine transmission of Theileria equi has been shown
in foal (Allsopp et al., 2007). Infection of horse fetus with
∗ Corresponding author. Tel.: +98 9163194663; fax: +98 6113360807.
E-mail addresses: m.nouri@scu.ac.ir, mnoori25@gmail.com (M. Nouri).
T. equi may lead to abortion, stillbirth, or the birth of live
foal with neonatal piroplasmosis (Allsopp et al., 2007;
Phipps and Otter, 2004). Vertical transmission of Theileria
sergenti in cows was confirmed by polymerase chain
reaction (PCR) (Baek et al., 2003).
The current study is the first report of the concurrent
isolation of T. lestoquardi from a ewe and her aborted fetus.
2. Materials and method
In July 2012, a mob of 450 ewes, in southwest of Iran,
was examined for approximately 35 late-gestation abor-
tions and 17 ewes’ mortality for a period of 5 days. A
case of recently aborted fetus with the respective ewe
was transported to the Ahvaz Veterinary Hospital for more
diagnostic evaluations. Depression and fever (41.8 ◦C) were
http://dx.doi.org/10.1016/j.vetpar.2014.04.007
0304-4017/© 2014 Elsevier B.V. All rights reserved.

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A. Zakian et al. / Veterinary Parasitology 203 (2014) 322–325 323
Fig. 1. The smears of peripheral blood (A and B) showing Theileria lestoquardi piroplasms ( ) in the red blood cells and Koch blue body in the lymphocytes
( ), and the impression smears (C and D) showing the ewe liver and the respective fetus spleen ( ); Giemsa stain. Ewe (B and C); fetus (A and D).
the most obvious clinical signs in animals in the cur-
rent study. The icteric mucosa, the presence of ticks on
the animal body, submandibular and subscapular lymph
node enlargement, weakness, increased respiration, and
pulse rates were also observed. The packed cell volume
(13%), red blood cell (RBC) concentration (4.41 × 103/␮l),
and hemoglobin (3.9 g/dl) of ewe were less than the lower
point of the normal range. Blood smears were prepared
using blood taken from jugular vein, left to dry and fixed
with methanol, and finally stained with the conventional
Giemsa stain (Baharafshan, Iran). Direct impression smears
were prepared from the dead ewe liver and aborted fetus
spleen, followed by staining procedures with the Giemsa
stain. Small pieces (1 cm × 1 cm × 1 cm) of liver and spleen
were cut and stored at −70 ◦C for subsequent DNA analysis.
The DNA was extracted from the liver and spleen using DNA
extraction kit (Roche, Germany) and the extracted DNA was
stored at −20 ◦C until subsequent analysis. Briefly, pieces
from each infected organ were cut (3 mm × 3 mm; weight
≈25 mg) and lysed in 100-␮l buffer followed by degra-
dation with 10-␮l proteinase K for 30 min at 55 ◦C. After
addition of 270-␮l binding buffer, the mixture was incu-
bated for 10 min at 70 ◦C before adding 320 ␮l of absolute
ethanol. The solution was briefly vortexed before transfer-
ring on to the column. The column was first centrifuged
and washed twice with 500-␮l washing buffer. Finally, DNA
was eluted from the carrier with elution buffer.
3. PCR amplification
In order to amplify the part of 18S rRNA gene from the T.
lestoquardi, primer sense (5 -CACAGGGAGGTAGTGACAAG)
and antisense (5 -CTAAGAATTTCACCTCTGACA) were used
(Zaeemi et al., 2011). The PCR was performed in final vol-
umes of 100 ␮l containing 100 ng of DNA template, 10 mM
Tris–HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 ␮M
(each) deoxynucleotide triphosphates (dNTPs), 0.4 ␮M of
each primer, and 2.5 U of Taq DNA polymerase (Invitro-
gen, Karlsruhe, Germany). The Bio-Rad Thermocycler was
programmed as follows: initial denaturation (95 ◦C, 2 min)
followed by PCR amplification with 35 cycles of 94 ◦C for
45 s, 56 ◦C for 45 s, 72 ◦C for 45 s, and a final extension of

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324 A. Zakian et al. / Veterinary Parasitology 203 (2014) 322–325
1 cycle at 72 ◦C for 10 min. Negative controls (containing
no DNA) were included in each PCR. PCR products were
then separated on 1.8% agarose gel, stained with ethidium
bromide, and observed under ultraviolet (UV) light. Sizes
were determined by comparison against the DNA length
standard marker (Invitrogen, Karlsruhe, Germany).
4. Purification of PCR products from agarose gel
The PCR product was isolated from the agarose gel using
the gel extraction kit (Fermentas, Lithuania) according to
the manufacturer’s instructions. Briefly, the DNA bands
were cut from the gel under UV illumination and dis-
solved in the binding buffer at 60 ◦C. The dissolved agarose
was transferred into the Fermentas column. After washing
twice with the washing buffer, the DNA bound to the carrier
was eluted using 20-␮l Tris–EDTA buffer.
5. DNA sequencing and sequence analysis
The purified PCR product was sequenced from both
ends using a dideoxy termination method in an Applied
Biosystems 373 DNA sequencer. The final sequence was
determined by using overlapping fragments. Database
nucleotide sequence homology searches were made using
the basic local alignment search tool (BLAST) pro-
gram (Altschul et al., 1990) from the NCBI GenBank®
(www.ncbi.nlm.nih.gov).
6. Results
Samples of the blood smears from fetus (A) and ewe
(B) showed piroplasma forms of Theileria in the RBC and
the Koch blue body in the circulatory lymphocytes (Fig. 1).
The impression smears from the liver of ewe (C) and the
spleen of her fetus (D) showed the Koch blue bodies in
the tissues (Fig. 1). The PCR performed using 18S RNA to
test for the presence of T. lestoquardi. DNA samples from
liver and spleen yielding a product of the expected size
(428 bp) were tentatively scored as positive for T. lesto-
quardi (Fig. 2). The negative-control reactions included
in each PCR run did not yield amplification products. To
confirm the presence of T. lestoquardi, the fragment was
purified and sequenced. To perform sequence comparison
of T. lestoquardi sequence and other ribosomal DNA (rDNA)
sequences, we searched the NCBI GenBank® with BLAST
by using the entire sequence as query. We found nine hits
with detectable sequence identity from the amplified PCR
products to T. lestoquardi (one hit with 100% identity and
eight hits with 99% identity). The results showed 100% and
99% identity to the registered sequence for T. lestoquardi
18S rDNA gene (JQ917458; it has recently been published
in database) and small-subunit rDNA gene (AF081135,
KC778786, KC778785, AJ006446, AY260185, AY260184,
AY260183, and KF771185), respectively.
7. Discussion
The previous report from the region confirmed T. lesto-
quardi as the main Theileria infection in sheep (Shayan
et al., 2011). For molecular confirmation, the sequence data
Fig. 2. Agarose gel electrophoresis of the PCR products of T. lestoquardi
18S rDNA gene isolated from the infected sheep and respective aborted
fetus. Each lane was loaded with 8 ␮l of the total reaction and the gel
was stained with ethidium bromide. Lane 1: the sheep liver; lane 2: the
respective fetus spleen; the negative control lane (Neg) was loaded with
water; marker lane (M) is shown with DNA size.
from the amplified product were applied for searching the
database using the NCBI tool. Comparison of the results
revealed that the nucleotide sequence of the amplified PCR
product was 100% identical to the aligned part of T. lesto-
quardi sequence in database.
The most economically known impact of the malignant
theileriosis in ewe is related to the high rate of mortal-
ity, and the disease-induced fever may contribute to the
occurrence of abortion. However, the evidence of transpla-
cental transmission of the parasite and expulsion of dead
fetus at term may suggest an active interaction of the
parasite with fetus before abortion. Previous reports con-
firmed the birth of calf (Baek et al., 2003) and foal (Allsopp
et al., 2007; Phipps and Otter, 2004) with theileriosis that
were infected through placenta. It assumes that virulence
of Theileria and host characteristics may affect the feto-
parasite interaction output. In the present clinical case,
after using anti-Theileria drug (Buparvaquone 5%, Butonix®,
Arfan daru, Tehran, Iran), the mortality and abortions were
ceased.
Finally, this is the report of vertical transfer of T. lesto-
quardi and its possible potential for inducing abortion
storm in ewe flock.
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