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Veterinary Parasitology 147 (2007) 161–165

Short communication

Molecular identification, genetic diversity and distribution of

Theileria and Babesia species infecting small ruminants§

Kursat Altay, Nazir Dumanli, Munir Aktas *

Department of Parasitology, Faculty of Veterinary Medicine, University of Firat, 23119 Elazig, Turkey

Received 22 December 2006; received in revised form 29 March 2007; accepted 3 April 2007

Abstract

Detection and identification of Theileria and Babesia species in 920 apparently healthy small ruminants in eastern Turkey, as

well as parasite genetic diversity, was investigated using a specifically designed reverse line blot (RLB) assay. The hypervariable V4

region of the 18S ribosomal RNA (rRNA) gene was amplified and hybridized to a membrane onto which catchall and species-

specific oligonucleotide probes were covalently linked. Three Theileria and one Babesia genotype were identified. Comparison of

the Theileria genotypes revealed 93.6–96.2% similarity among their 18S rRNA genes. Two Theileria shared 100% and 99.7%

similarity with the previously described sequences of T. ovis and Theileria sp. OT3, respectively. A third Theileria genotype was

found to be clearly different from previously described Theileria species. The genotype was provisionally designated as Theileria

sp. MK. The Babesia genotype shared 100% similarity with Babesia ovis. The survey indicated a high prevalence of piroplasm

infections in small ruminants (38.36%). Theileria spp. prevalence was 36.08%. Prevalence of B. ovis was 5.43%. The most abundant

Theileria species identified was T. ovis (34.56%) followed by Theileia sp. MK (1.30%) and Theileria sp. OT3 (0.43%).

# 2007 Elsevier B.V. All rights reserved.

Keywords: Theileria; Babesia; PCR; Microscopy; Sheep; Goats

1. Introduction

Piroplasmosis is caused by the tick-borne hemopro-

tozoan Theileria and Babesia and has a major impact on

livestock production in tropical and subtropical areas of

the world. Babesia ovis, B. motasi and B. crassa are

species that cause small ruminant babesiosis, whereas

ovine and caprine theileriosis is caused by Theileria

lestoquardi (T. hirci), T. ovis, T. separata, Theileria sp.

China and T. recondita (Ahmed et al., 2006). Recently,

two ovine Theileria genotypes, Theileria sp. OT1 and

§ Note: Nucleotide sequence data reported in this paper are available

in GenBank, EMBL and DDBJ databases under accession numbers

from EF092452 to EF092456.

* Corresponding author. Tel.: +90 424 237 0000;

fax: +90 424 238 8173.

E-mail address: maktas@firat.edu.tr (M. Aktas).

0304-4017/$ – see front matter # 2007 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetpar.2007.04.001

Theileria sp. OT3, have been described (Nagore et al.,

2004).

Babesia ovis, T. lestoquardi and Theileria sp. China

are highly pathogenic especially to sheep and cause

severe clinical infections. The other species are less

pathogenic or non-pathogenic in small ruminants

(Friedhoff, 1997). In acute cases, small-ruminant

piroplasmosis can be diagnosed by microscopic

examination of Giemsa-stained thin blood smears and

by clinical symptoms. But, following acute infections,

recovered animals frequently retain subclinical infec-

tions. Serological methods are employed in diagnosing

subclinical infections, but false positive and false

negative results are commonly observed due to cross-

reactions. Therefore, a highly specific and sensitive

method for the diagnosis of piroplasms is required.

Recently, species-specific polymerase chain reaction

(PCR) and PCR-based reverse line blot (RLB)

K. Altay et al. / Veterinary Parasitology 147 (2007) 161–165162

hybridization methods have been developed and used

(Schnittger et al., 2004; Aktas et al., 2005, 2007).

The present study explored the use of RLB assay to

improve detection and identification of Theileria and

Babesia species infecting sheep and goats. A pre-

liminary survey suggested the presence of novel

Theileria genotype.

2. Materials and methods

The study was carried out between June 2005 and

October 2006 in 10 provinces of eastern Turkey. Whole

blood samples were collected from 705 sheep and 215

goats. All were clinically healthy. A thin smear was

prepared and numbered in the field for each sample by

the same person for the microscopic examination.

Approximately 20,000 erythrocytes per slide were

examined and the percent infected calculated.

DNA extraction was performed essentially as

described by d’Oliveira et al. (1995). For the

amplification of Theileria and Babesia, one set of

primers was used to amplify approximately �390

and �430 bp fragments of the hypervariable V4

region of the 18S rRNA gene. The forward [RLB-F2

(50-GACACAGGGAGGTAGTGACAAG-30)] and the

reverse [RLB-R2 (Biotin-50-CTAAGAATTTCACCT-

CTGACAGT-30)] primers are as described previously

(Georges et al., 2001). The PCR volume and reaction

conditions applied were similar to those described by

Nagore et al. (2004). Preparation, hybridization and

stripping of the RLB membrane were performed as

outlined by Gubbels et al. (1999).

All the oligonucleotid probes, with the exception of a

new probe (50-CATTGTTTCTTCTCATGTC-30)designed and used for the first time in this study, were

previously tested against Theileria and Babesia and

gave positive results for the corresponding species

(Gubbels et al., 1999; Nagore et al., 2004; Schnittger

et al., 2004). The specificity of the new probe was also

tested against different PCR products corresponding to

the hypervariable V4 region of Theileria and Babesia

species.

After amplification of the hypervariable V4 region of

the 18S rRNA gene with primer pair RLB-F/RLB-R

(Gubbels et al., 1999), generated DNA fragments, of

approximately 460 and 520 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. The partial sequences of

the 18S rRNA genes determined in this study for T. ovis,

B. ovis, Theileria sp. OT3 and Theileria sp. MK, were

deposited in the EMBL/GenBank databases under

accession numbers from EF092452 to EF092456. Each

construct was sequenced at least three times and

subjected to BLAST similarity searches. The multiple

sequence alignments were performed using the Jalview

program (Clamp et al., 2004), based on Clustal W

(Thompson et al., 1994). A phylogenetic tree was

created from the sequences of the 18S rRNA genes of

the small ruminant main Theileria and Babesia isolates

available from GenBank and the novel sequences

described here, using the neighbor-joining method in

MEGA Version 3.1 (Kumar et al., 2004).

3. Results

Primers RLB-F2 and RLB-R2 amplified bands of

approximately�390 and �430 bp corresponding to the

hypervariable V4 region of Theileria and Babesia

species (data not shown). These PCR products were

hybridized onto the membrane and were observed for

reaction to specific oligonucleotide probes. All the PCR

positive samples showed positive reactions with their

corresponding specific probes. However, 12 samples

gave positive signals to catchall and Theileria genera-

specific probes, but did not show any reaction with the

other species-specific probes tested. This indicated the

presence of a novel Theileria genotype. One repre-

sentative sample of the products was sequenced and

used to design a new probe to enhance the assay

identification capability. The new probe gave a positive

reaction with its corresponding genotype and did not

cross-react with other Theileria and Babesia species

tested. PCR performed on uninfected sheep DNA did

not yield any detectable product on agarose gel. As

expected, cross-reaction between T. lestoquardi and T.

annulata was observed.

In this study, four genotypes, three Theileria and one

Babesia, were identified (Table 1). Comparison of the

partial sequences of 18S rRNA gene revealed 93.6–

96.2% homology among the three Theileria genotypes,

two of which shared 100% and 99.7% identity with the

recently reported sequences for the 18S rRNA genes of

T. ovis and Theileria sp. OT3 Spain. Therefore, they

were classified and named as T. ovis and Theileria sp.

OT3. The third was 93.3% similar to Theileria sp. OT3

Spain and 96.1% similar to T. ovis Turkey. This third

genotype also differed clearly from all the Theileria

species currently available in the GenBank database and

showed only 96.9% similarity with the most closely

corresponding BLAST species, Theileria sp. China

(cattle). Thus, it represented a novel Theileria genotype,

and was provisionally designated Theileria sp. MK.

K. Altay et al. / Veterinary Parasitology 147 (2007) 161–165 163

Table 1

Percent identity of the main Theileria and Babesia species discussed by Clustal W

Species 1 2 3 4 5 6 7 8 9 10 11 12 13

T. ovis (EF092453)a 1 100 100 94.9 96.2 94.9 94.5 94.7 95.3 94.7 94.3 95.8 81.8 81.8

T. ovis Turkey (AY508460) 2 100 94.9 96.1 97.1 96.7 96.9 97.3 96.6 96.8 97.4 81.8 87.4

Theileria sp. OT3 (EF092455)a 3 100 93.6 94.9 99.7 94.9 93.1 92.9 93.3 93.3 79.2 79.2

Theileria sp. MK (EF092456)a 4 100 96.1 93.3 95.6 93.4 94.6 96.9 93.7 81.1 81.3

Theileria sp. China 1 (AF081136) 5 100 96.7 99.4 96.0 96.2 97.0 96.2 79.5 86.7

Theileria sp. OT3 Spain (AY533145) 6 100 96.7 96.5 96.3 96.8 96.0 79.1 86.3

Theileria sp. OT1 (AY533143) 7 100 96.1 96.3 97.1 96.1 80.2 87.0

T. lestoquardi (AF081135) 8 100 96.3 96.8 99.2 82.9 87.4

T. separata (AY260175) 9 100 96.9 95.1 81.9 87.2

Theileria sp. China (cattle) (AF036336) 10 100 96.2 81.8 87.6

T. annulata (AY508464) 11 100 82.7 87.1

B. ovis (EF092454)a 12 100 100

B. ovis Spain (AY533146) 13 100

a Sequences described in this study.

This genotype showed 93.4%, 93.7%, 94.6%, 96.1%

and 95.6% similarity with T. lestoquardi, T. annulata, T.

separata, Theileria sp. China 1 and Theileria sp. OT1,

respectively. The single Babesia genotype in this study

showed 100% similarity to B. ovis and was therefore

identified as B. ovis.

Phylogenetic analysis showed evidence of two

distinct groups of small ruminant piroplasms (Fig. 1).

Group one comprised the Babesia sequences, which

were divided into three monophyletic clades, one

consisting of B. ovis and the others B. crassa and B.

motasi, with well-supported separation among these

species. The B. ovis sequence described in this study

formed a well-supported clade (100%) with the other B.

ovis sequences, clearly distinct from B. crassa and B.

motasi. Group two comprised Theileria species. The T.

ovis and Theileria sp. OT3 sequences described in this

study formed a well-supported clade with the corre-

sponding sequences of T. ovis and Theileria sp. OT3,

whereas Theileria sp. MK belonged to a different clade.

However, the position of the cluster formed by Theileria

sp. MK was related to T. ovis and Theileria sp. OT1

clades.

Thin blood smears revealed parasitemia in infected

animals ranging from 0.01% to 1%. Piroplasms,

detected inside erythrocytes, were polymorphous and

comprised: round, oval, ring, anaplasmoid (Theileria

spp.) or double pyriform and single ring (Babesia spp.)

forms.

Prevalence of each piroplasm species identified in

sheep and goats is shown in Table 2. Overall prevalence

of piroplasms was estimated as 38.36% (353/920) by

RLB. Theileria spp. prevalence was 36.08% (332/920),

whereas prevalence of B. ovis was 5.43% (50/920). The

most abundant Theileria species identified was T. ovis

(318/920, 34.56%) followed by Theileia sp. MK (12/

920, 1.30%). Theileria sp. OT3 occurred in only four

samples (4/920, 0.43%). Single infection by T. ovis was

detected in 287 samples. Mixed infections were

observed in 31 samples. One animal infected with T.

ovis was also infected with Theileria sp. OT3 and

Theileria sp. MK. Of the 50 animals infected with B.

ovis, 29 were also infected with T. ovis.

4. Discussion

Parasite species identification using conventional

methods is difficult, particularly when mixed infections

occur. Conversely, PCR-based molecular genetic

techniques allow sensitive detection of specific pir-

oplasms. The RLB assay is a powerful tool and practical

assay, since it is able to detect extremely low

parasitemia rates and simultaneously identify Theileria

and Babesia species (Gubbels et al., 1999; Schnittger

et al., 2004). This is the first report in which RLB has

been used to detect and identify ovine Theileria and

Babesia parasites in Turkey. The survey identified new

18S rRNA gene sequences and a novel Theileria

genotype, and therefore contributed to better insight

into small ruminant piroplasm distribution and their

phylogenetic diversity.

The oligonucleotide probes used in this study reacted

with their corresponding species or strain and did not

cross-react, except the T. lestoquardi specific probe

which did cross-react with T. annulata. The result

agrees with a previous report (Nagore et al., 2004). If

PCR products hybridize only to the catchall or genus

probes, it may indicate the presence of a novel genotype

or variant of a known species (Gubbels et al., 1999;

Schnittger et al., 2004). In this study, 12 samples gave

K. Altay et al. / Veterinary Parasitology 147 (2007) 161–165164

Fig. 1. Neighbor-joining analysis of the 18S rRNA gene of the small ruminant 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

package. The GenBank accession numbers are in parentheses. Sequences described in this study are in bold. Scale bar represents nucleotide

substitutions per position.

Table 2

Distribution and frequency of Theileria and Babesia infections detected by reverse line blot (n = 920)

Parasite types

Piroplasm Theileria spp. T. ovis Theileria sp. MK Theileria sp. OT3 B. ovis

287 287 287 – – –

29 29 29 – – 29

21 – – – – 21

11 11 – 11 – –

3 3 – – 3 –

1 1 1 1 – –

1 1 1 – 1 –

Total (%) 353 (38.36) 332 (36.08) 318 (34.56) 12 (1.30) 4 (0.43) 50 (5.43)

K. Altay et al. / Veterinary Parasitology 147 (2007) 161–165 165

positive reactions with catchall and generic Theileria

probes, but they did not show a reaction with the other

species-specific oligonucleotide probes tested. Thus, a

new probe was designed from the representative

sequence and included in the assay. The probe was

also tested against ovine and non-ovine Theileria and

Babesia species, and cross-reactions were not observed.

Sequence comparisons and phylogenetic results

documented in this study demonstrated that at least

three genetically distinct Theileria and one Babesia

genotype were present in small ruminants in Turkey.

The presence of T. ovis and B. ovis were expected, since

these parasites have been reported previously (Aktas

et al., 2005; Altay et al., 2005). However, Theileria sp.

OT3 described and first reported in Spain (Nagore et al.,

2004) and Theileria sp. MK was detected for the first

time in Turkey. Phylogenetic analysis provided evi-

dence that Theileria sp. MK is distinct from previously

described Theileria species. These results indicate that

Theileria genotypes in small ruminants comprise a

heterologous group, not restricted to Theileria sp. OT1

and OT3 (Nagore et al., 2004), with more genotypes,

such as the novel Theileria sp. MK described here.

As Theileria sp. MK was identified in sheep and

goats, it is assumed that the host of the genotype is the

small ruminant. It is noteworthy that the 18S rRNA gene

sequence of the parasite is different from that of

Theileria sp. China 1 (96.1% similarity) and T.

lestoquardi (93.4% similarity) which are pathogenic

for small ruminants. As the genotype was detected in

apparently healthy animals, it seems to be non-

pathogenic. However, an interesting finding was the

high parasitemia rates observed in blood smears

infected with the genotype when compared with T.

ovis and Theileria sp. OT3 (data not shown).

In conclusion, this study has revealed three Theileria

(T. ovis, Theileria sp. OT3, Theileria sp. MK) and one

Babesia (B. ovis) parasite. The RLB performed has

revealed a novel Theileria genotype infecting sheep and

goats. The assay provided more accurate data on

prevalence of infection and allowed direct identification

of species and mixed infections.

Acknowledgements

This work was supported financially by a grant (104

O 393) from the Scientific and Technical Research

Council of Turkey (TUBITAK). The authors wish to

thank Dr. Ana Hurtada (Department of Animal Health,

Instituto Vasco de Investigacion y Desarrollo Agrario

Berreaga, Bizkaia, Spain) for the Theileria sp. OT1,

OT3 and B. motasi DNAs; Dr. Jabbar Ahmed

(Department of Immunology and Cell Biology, Borstel,

Germany) for the T. lestoquardi DNA; Dr. Dirk Geysen

(Department of Animal Health, Institute for Tropical

Medicine, Antwerp, Belgium) for the T. parva DNA.

We are grateful to Dr. Anil Ica (University of Erciyes,

Faculty of Veterinary Medicine, Kayseri, Turkey) for

helping in setting up the RLB assay.

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