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Short communication First molecular diagnosis of Babesia vogeli in domestic dogs from Turkey Aynur Gu ¨lanber a, * , Andre ´ Gorenflot b , Theo P.M. Schetters b,c , Bernard Carcy b a Istanbul University, Faculty of Veterinary Medicine, Parasitology Department, 34320-Avcılar, Istanbul, Turkey b Laboratoire de Biologie Cellulaire et Mole ´culaire, ERT 1038 ‘‘Vaccination antiparasitaire’’, UFR des Sciences Pharmaceutiques et Biologiques, 15 avenue Charles Flahault, BP 14491, F-34093 Montpellier cedex 5, France c Department of Parasitology, Intervet International B.V., Postbus 31, 5830 AA Boxmeer, Netherlands Received 6 October 2005; received in revised form 23 February 2006; accepted 24 February 2006 Abstract Microscopic examination of Giemsa-stained peripheral blood smears collected from three naturally infected dogs originating from Turkey revealed the presence of large (around 4.5–5.0 mm) intraerythrocytic Babesia parasites in all dogs. DNA was extracted from the three infected blood samples and an around 410 bp portion of the 18 S rDNA gene of Babesia species was PCR amplified for subsequent molecular characterization. RFLP analysis of the PCR products suggested the presence of the species B. vogeli in all infected dogs and sequencing of the PCR products from two of the three samples revealed 100% identity among the two Turkish isolates. Comparisons with the equivalent 410 bp portions of the 18 S rDNA gene of Babesia species confirmed the affiliation of these isolates to the B. vogeli species. This is the first report and molecular characterization of dog infection with a large Babesia species in Turkey. # 2006 Elsevier B.V. All rights reserved. Keywords: Babesiosis; Babesia vogeli; ssu-rDNA gene; Dog; Turkey 1. Introduction Canine babesiosis is characterized by fever, anaemia and haemoglobinuria and is generally attributed to the tick-transmitted intracellular haemo- protozoan parasites Babesia canis (Piana and Galli- Valerio, 1895) and Babesia gibsoni (Patton, 1910). These two canine Babesia species are morphologi- cally distinguishable since they correspond to large (4–5 mm) and small piroplasm (1.5–2.5 mm), respec- tively (Kuttler, 1988). Thus, on the basis of Giemsa- stained peripheral blood smear examination, large and small canine Babesia isolates collected around the world have been generally attributed to B. canis or B. www.elsevier.com/locate/vetpar Veterinary Parasitology 139 (2006) 224–230 * Corresponding author. Tel.: +90 212 473 70 70; fax: +90 212 473 72 41. E-mail address: [email protected] (A. Gu ¨lanber). 0304-4017/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2006.02.035

First molecular diagnosis of Babesia vogeli in domestic dogs from Turkey

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Short communication

First molecular diagnosis of Babesia vogeli

in domestic dogs from Turkey

Aynur Gulanber a,*, Andre Gorenflot b,Theo P.M. Schetters b,c, Bernard Carcy b

a Istanbul University, Faculty of Veterinary Medicine, Parasitology Department, 34320-Avcılar, Istanbul, Turkeyb Laboratoire de Biologie Cellulaire et Moleculaire, ERT 1038 ‘‘Vaccination antiparasitaire’’,

UFR des Sciences Pharmaceutiques et Biologiques, 15 avenue Charles Flahault,

BP 14491, F-34093 Montpellier cedex 5, Francec Department of Parasitology, Intervet International B.V., Postbus 31, 5830 AA Boxmeer, Netherlands

Received 6 October 2005; received in revised form 23 February 2006; accepted 24 February 2006

Abstract

Microscopic examination of Giemsa-stained peripheral blood smears collected from three naturally infected dogs originating

from Turkey revealed the presence of large (around 4.5–5.0 mm) intraerythrocytic Babesia parasites in all dogs. DNA was

extracted from the three infected blood samples and an around 410 bp portion of the 18 S rDNA gene of Babesia species was

PCR amplified for subsequent molecular characterization. RFLP analysis of the PCR products suggested the presence of the

species B. vogeli in all infected dogs and sequencing of the PCR products from two of the three samples revealed 100% identity

among the two Turkish isolates. Comparisons with the equivalent 410 bp portions of the 18 S rDNA gene of Babesia species

confirmed the affiliation of these isolates to the B. vogeli species. This is the first report and molecular characterization of dog

infection with a large Babesia species in Turkey.

# 2006 Elsevier B.V. All rights reserved.

Keywords: Babesiosis; Babesia vogeli; ssu-rDNA gene; Dog; Turkey

www.elsevier.com/locate/vetpar

Veterinary Parasitology 139 (2006) 224–230

1. Introduction

Canine babesiosis is characterized by fever,

anaemia and haemoglobinuria and is generally

attributed to the tick-transmitted intracellular haemo-

* Corresponding author. Tel.: +90 212 473 70 70;

fax: +90 212 473 72 41.

E-mail address: [email protected] (A. Gulanber).

0304-4017/$ – see front matter # 2006 Elsevier B.V. All rights reserved

doi:10.1016/j.vetpar.2006.02.035

protozoan parasites Babesia canis (Piana and Galli-

Valerio, 1895) and Babesia gibsoni (Patton, 1910).

These two canine Babesia species are morphologi-

cally distinguishable since they correspond to large

(4–5 mm) and small piroplasm (1.5–2.5 mm), respec-

tively (Kuttler, 1988). Thus, on the basis of Giemsa-

stained peripheral blood smear examination, large and

small canine Babesia isolates collected around the

world have been generally attributed to B. canis or B.

.

A. Gulanber et al. / Veterinary Parasitology 139 (2006) 224–230 225

gibsoni. However, the development of molecular

biological methods (Zahler et al., 1998; Carret et al.,

1999; Caccio et al., 2002; Birkenheuer et al., 2003;

Matjila et al., 2004) for the characterization of canine

Babesia isolates collected around the world sug-

gested that the taxonomy of Babesia species

infecting dogs is more complicated than those

initially described. As an example, the molecular

characterization of small canine Babesia isolates

revealed the existence of taxonomic entities attrib-

uted (Kjemtrup et al., 2000; Zahler et al., 2000a) or

not (Zahler et al., 2000b; Camacho et al., 2001) to B.

gibsoni. In the case of large Babesia isolates, it has

been initially proposed that they are separated into

three subspecies with different vector specificity and

prevalence: B. canis canis is transmitted by

Dermacentor reticulatus in Europe, B. canis vogeli

by Rhipicephalus sanguineus in Southern Europe

and North Africa and B. canis rossi by Haemophy-

salis leachi in South Africa (Uilenberg et al., 1989).

Molecular methods confirmed three distinct taxo-

nomic entities for isolates usually attributed to B.

canis (Zahler et al., 1998; Carret et al., 1999; Caccio

et al., 2002). However, a new taxonomic entity which

is not attributed to this species was recently

discovered (Birkenheuer et al., 2004). It has been

suggested that these taxonomic entities might

correspond to genetically distantly related species,

initiating discussion about classification of canine

Babesia species. This is not the aim of the present

paper and we will adopt here the nomenclature B.

canis, Babesia rossi and Babesia vogeli as it

becomes increasingly used in the literature (Depoix

et al., 2002; Schetters, 2005; Passos et al., 2005). In

addition to their ability to unambigously characterize

large Babesia isolates, these molecular methods gave

a better understanding of the distribution of these

three taxonomic entities around the world. They

confirmed the prevalence of B. canis in Europe

(Zahler et al., 1998; Carret et al., 1999, Caccio et al.,

Table 1

Clinical features and results of haematological testing for three domestic

Breed Sex Age Clinical signs RBC � 1

Golden M 0.5 Lethargy, fever, less of weight 1.97

Boxer M 1.5 Lethargy, fever, less of weight 3.62

Boxer F 1 Lethargy, fever, less of weight 3.04

2002; Criado-Fornelio et al., 2003; Duh et al., 2004;

Foldvari et al., 2005; Matjila et al., 2005). Similarly,

the prevalence of B. rossi was confirmed in South

Africa (Zahler et al., 1998; Carret et al., 1999), and

this species was also recently detected in Eastern

Sudan (Oyamada et al., 2005). Surprisingly, result

showed that B. vogeli is more world-widespread than

previously described. As expected, it was detected in

Europe and North Africa (Zahler et al., 1998; Carret

et al., 1999; Caccio et al., 2002; Criado-Fornelio

et al., 2003; Duh et al., 2004), but additional studies

revealed that the parasite was also found in Northern

and Southern America (Birkenheuer et al., 2003;

Passos et al., 2005), Australia (Jefferies et al., 2003),

Japan (Inokuma et al., 2004) and Eastern and South

Africa (Matjila et al., 2004; Oyamada et al., 2005).

Knowledge of the prevalence of B. canis, B. vogeli

and B. rossi (or of new taxonomic entities) around

the world is of epidemiological interest. It might be

also of particular interest for efficient therapy against

canine babesiosis as clinical signs for the develop-

ment of disease in infected dogs are distinct between

B. canis, B. vogeli and B. rossi isolates (Uilenberg

et al., 1989; Schetters et al., 1997), which might

affect the rationale of vaccine development (Schet-

ters, 2005).

The epidemiological aspect of canine babesiosis

and classification of Babesia isolates infecting dogs

in Turkey are still unknown. In the present study,

we report the first B. vogeli infection in domestic

dogs living in Istanbul using molecular biological

techniques.

2. Materials and methods

Three domestic dogs living in Istanbul (Table 1) and

with clinical manifestations suspicious of babesiosis

(i.e., complaints of lethargy, 40 8C fever and loss of

weight) were brought to the Veterinary Faculty Clinics

dogs from Turkey infected with large Babesia parasites

05/ml (5.5–8.5) Hct % (37–55) Hb dl (12–18) Sample

12 4.3 Turk1

24 8.2 Turk2

21.3 7.3 Turk3

A. Gulanber et al. / Veterinary Parasitology 139 (2006) 224–230226

ig. 1. Detection of pear-shaped large Babesia parasites in

e peripheral blood of infected dogs from Turkey. Blood smear

as Giemsa-stained and observed under light microscopy

magnification � 1000).

of Istanbul (dogs 1–2 in June 2005 and dog 3 in

September 2002). These dogs were reported to be

heavily infested with ticks in the past, but no ticks were

found during the physical examination. Peripheral

blood samples (of dogs 1–3, hereafter designated

Turk1, Turk2 and Turk3, respectively) were collected

for immediate complete blood count, biochemical

analysis and microscopic examination. Giemsa-stained

blood smears were made and examined under the light

microscope (magnification � 1000) for direct detection

of intraerythrocytic stages. Blood samples from

infected dogs were collected in EDTA tubes and the

DNA was extracted from 200-ml aliquots of EDTA

blood using the NucleoSpinR Blood QuickPure Kit as

recommended by the manufacturer (Macherey-Nagel,

Hoerdt, France). Molecular characterization of the

Turk1, Turk2 and Turk3 samples was firstly performed

by a PCR-RFLP analysis of an around 410 bp portion of

the 18 S rDNA genes according to Carret et al. (1999).

For comparison, the PCR-RFLP analysis was also

conducted with genomic DNA extracted from the in

vitro cultures of B. canis and B. rossi isolates which

have served as reference isolates for the elaboration of

this molecular diagnosis (Carret et al., 1999). Briefly,

PCR amplifications were carried out with the PCR

primers Piro-A (50-AAT TAC CCA ATC CTG ACA

CAG GG-30) and Piro-B (50-TTA AATACG AAT GCC

CCC AAC-30) (Olmeda et al., 1997). As control, a PCR

amplification was performed with genomic DNA

extracted from a blood sample collected on a 2-year-

old female beagle dog without history of babesiosis and

living in a confined environment. For subsequent RFLP

analysis of the Piro-A/Piro-B products, 1/5 of each

amplification product was digested for 3 h respectively

with 10 U of HinfI and TaqI restriction enzymes in their

appropriate buffer (Life Technologies, United King-

dom). Results of these digestions were analyzed on an

ethidium bromide stained 2% Seakem agarose gel

(FMC Bioproducts, USA). The molecular character-

ization of canine babesiosis samples from Turkey was

certified by sequencing the Piro-A/Piro-B amplification

products from the Turk1 and Turk2 samples. They were

subcloned in the TOPO1 vector using the TOPO TA

cloning1 kit as recommended by the manufacturer

(Invitrogen, USA) and the sequencing reaction was

performed on both strands by Genome express S.A.

(Grenoble, France) by using the dideoxychain-termina-

tion method (Sanger et al., 1977). The nucleotide

sequences are available in GenBank, EMBL, and DDBJ

databases under the accession numbers AM183215

(Turk1) and AM183216 (Turk2). The multiple

sequence alignments of Piro-A/Piro-B region of B.

vogeli 18 S rDNA sequences were performed using the

Clustal w program (Thompson et al., 1994). The Piro-

A/Piro-B region of B. vogeli 18 S rDNA sequences

from Africa (Egypt 1: AY371197; Eastern Sudan:

DQ111766; South Africa: AF548006), Australia

(AY102162), Japan (AY0077719), America (Brazil:

AY371196; USA: AY371198) and Europe (France:

AY072925; Spain: AY150061) were included for

comparison.

3. Results and discussion

Three domestic dogs from Istanbul were pre-

sented at the clinic with signs of babesiosis.

Haematological examination revealed anaemia,

low haematocrit and haemoglobinemia (Table 1),

and Giemsa-stained blood smears showed the

presence of large (size of around 4.5–5.0 mm)

intraerythrocytic pear-shaped parasites (Fig. 1) in

all three dogs (estimated parasitaemia of 1.5%). As it

was the first diagnosis of canine babesiosis due to

large Babesia parasites in Turkey, we decided to use

molecular biological tools to further characterise the

parasites. Large canine Babesia isolates are basically

separated into three taxonomic entities (B. canis,

F

th

w

(

A. Gulanber et al. / Veterinary Parasitology 139 (2006) 224–230 227

B. vogeli and B. rossi) and different molecular

biological methods, in most cases based on the

amplification, sequencing and comparisons of the

18 S rDNA genes, have been developed for their

simple and sensitive detection (Zahler et al., 1998;

Carret et al., 1999; Caccio et al., 2002; Matjila et al.,

2004; Martin et al., 2006). The PCR-RFLP test

described by Carret et al. (1999) and sequencing of

partial 18 S rDNA gene can be used to distinguish

the three large canine Babesia species. The assay is

based on the polymorphism of TaqI and HinfI

restriction sites within a 407 bp fragment of the 18 S

rDNA genes from B. canis (no TaqI or HinfI

restriction site), B. vogeli (two TaqI but no HinfI

restriction sites) and B. rossi (one HinfI but no TaqI

restriction site). As analysis of the three isolates

(Turk1, Turk2 and Turk3) by this approach gave

identical results, only those obtained with isolate

Turk3 are presented in Figure 2. In agreement with a

canine Babesia molecular diagnosis, an expected

410 bp fragment from the 18 S rDNA gene was PCR

amplified from Turk3 with the Piro-A/Piro-B

primers set (Fig. 2A). These primers were designed

to specifically hybridize to Babesia 18 S rDNA

genes (Olmeda et al., 1997) and no signal of

amplification was observed with genomic DNA from

a control dog (Fig. 2A). The Piro-A/Piro-B PCR

fragment from Turk3 was not digested using HinfI as

restriction enzyme, but digestion with TaqI revealed

Fig. 2. PCR-RFLP analysis of Piro-A/Piro-B region of the small 18 S rDN

Piro-A/Piro-B regions from B. canis, B. rossi, canine blood samples T

electrophoresed in 0.8% Seakem agarose gel and stained by ethidium brom

and the canine blood sample Turk3 were then digested with TaqI (left) and H

Seakem agarose. Sizes of Piro-A/Piro-B amplification products and diges

two ethidium bromide-stained fragments with a size

of around 210 and 170 bp (the third expected one of

around 30 bp being not detectable), which indicated

that the parasite was B. vogeli. As controls for the

proper action of HinfI and for a B. rossi diagnosis,

the Piro-A/Piro-B fragment from B. rossi was

digested by this enzyme into two ethidium bro-

mide-stained fragments with a size of around 230

and 180 bp, but it was not digested by TaqI (Fig. 2B).

As expected, the Piro-A/Piro-B PCR fragment from

B. canis was neither digested by HinfI or TaqI

(Fig. 2B). To certify an infection of domestic dogs

from Turkey with B. vogeli, the entire 407 bp Piro-

A/Piro-B 18 S rDNA-amplicons from isolates Turk1

and Turk2 were sequenced. Sequencing demon-

strated 100% identity to each other and Blast-n

search confirmed that these two isolates were B.

vogeli. Comparison of these sequences with avail-

able Piro-A/Piro-B 18 S rDNA-derived sequences of

B. vogeli isolates originating from the five continents

indicates 100% identity with the B. vogeli sequence

from Egypt (Egypt1: accession number AY371197),

i.e., the reference isolate that had served for the

description of the taxon vogeli (Uilenberg et al.,

1989). Identities of at least 99% were found with

other isolates (1–4 substitutions), despite a very

distant geographical origin for some of them

(Table 2). As previously suggested by Passos

et al. (2005), such a conservation of sequences

A gene from the large Babesia parasite infecting dogs in Turkey. (A)

urk3 from Turkey and from a control dog were PCR amplified,

ide. (B) Piro-A/Piro-B amplification products from B. canis, B. rossi

infI (right) restriction enzymes. The gel was electrophoresed on 2%

ted products are indicated in base pair (bp).

A. Gulanber et al. / Veterinary Parasitology 139 (2006) 224–230228

Table 2

Polymorphic position and TaqI restriction sites position within Piro-A/Piro-B regions of B. vogeli 18 S rDNA sequences

Genotype (Gen Bank accession number) Polymorphic position Taql position

59 86 183 197 230 267 208–211 234–238

Turkey (AMI83215) – A A C C T + +

Africa

Egypt (AY371197) – * * * * * + +

Eastern Sudan (DQ111766) – * * T T n.d. + +

South Africa (AF548006) G * G T T * + +

Oceania

Australia (AY102162) – * * T T * + +

Asia

Japan (AY0077719) – * * T T * + +

America

Brazil (AY371196) – * * T T * + +

USA (AY371198) – C * T T * + +

Europe

France (AY072925) – C * T T * + +

Spain (AY150061) – C * T T A + +

Base numbering of polymorphic or TaqI position is according to the 407 bp genotype from Turkey. The B. vogeli 18 S rDNA sequence from

Turkey was compared with the available equivalent 407 bp sequences (i.e., corresponding to the 373–780 base position of genotype from Brazil)

from world-widespread B. vogeli isolates. Exceptions concern genotypes from South Africa and Australia, where comparisons were performed

with 355 and 363 bp available sequences in that area, respectively. (�), deletion; (*), same base as in genotype Turkey; (n.d.), not determined, (+)

presence of a TaqI restriction site.

between geographically distant isolates indicated

that all these world-widespread parasites infecting

dogs and attributed to B. vogeli belong to the same

taxonomic entity. On the basis of comparisons of

available partial or complete 18 S rDNA sequences,

Passos et al. (2005) previously described 13

polymorphic positions within the B. vogeli 18 S

rDNA gene. Six of these polymorphic positions

localized in the Piro-A/Piro-B region (i.e., between

nucleotide positions 373–780 according the com-

plete 18 S rDNA sequence from Brazil with

accession number AY371194) (Table 2). The most

notable polymorphism in the Piro-A/Piro-B 18 S

rDNA-derived sequences from Turkey (or Egypt)

and all other available equivalent sequences are the

positions 197 and 230 where the conserved T is

substituted for C in Turkey and Egypt1 (Table 2).

These substitutions also occurred in the sequence

Egypt2 (accession number AJ009796) (not shown).

Moreover, comparisons of available B. vogeli Piro-

A/Piro-B 18 S rDNA-derived sequences revealed an

extreme conservation of the two TaqI restriction sites

(positions 208–211 and 234–238) and that none of

the six polymorphic positions altered the TaqI

restriction map (Table 2). Although sequencing

remains the method of choice for the unambigous

molecular characterization of canine Babesia iso-

lates, this extreme conservation of TaqI restriction

map within B. vogeli Piro-A/Piro-B 18 S rDNA-

derived sequence further corroborates that unse-

quenced Turk3 isolate is B. vogeli. Finally, these

results indicate that the PCR-RFLP test developed

by Carret et al. (1999) is a valuable molecular

method for the epidemiological study of B. vogeli.

In conclusion, this study describes the first cases of

canine babesiosis due to a large Babesia species in

domestic dogs from Turkey. The high genetic

homology of the Piro-A/Piro-B 18 S rDNA-derived

sequences from the samples Turk1 and Turk2 with

sequences of B. vogeli indicates that at least in the area

of Istanbul this is the predominant species infecting

dogs. Moreover, the distribution of the tick vector that

transmits B. vogeli (Rhipicephalus sanguineus is

prevalent between 50 8N and 35 8S; Estrada-Pena

A. Gulanber et al. / Veterinary Parasitology 139 (2006) 224–230 229

et al., 2004) favours this possibility. For a better

understanding of transmission of canine babesiosis in

Turkey epidemiological studies on the prevalence of B.

vogeli parasites and R. sanguineus tick are necessary.

Acknowledgements

The authors would like to thank S. Randazzo and C.

Roques for helpful technical assistance. This work

was supported by The Research Fund of The

University of Istanbul (Project number: UDP-385/

20082004) and by a grant from Intervet International

(The Netherland).

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