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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).
References
Birkenheuer, A.J., Levy, M.G., Breitschwerdt, E.B., 2003. Devel-
opment and evaluation of a seminested PCR for detection and
differentiation of Babesia gibsoni (Asian genotype) and B. canis
DNA in canine blood samples. J. Clin. Microbiol. 41, 4172–
4177.
Birkenheuer, A.J., Neel, J., Ruslander, D., Levy, M.G., Breitsch-
werdt, E.B., 2004. Detection and molecular characterization of a
novel large Babesia species in a dog. Vet. Parasitol. 124, 151–
160.
Caccio, S.M., Antunovic, B., Moretti, A., Mangili, V., Marinculic,
A., Baric, R.R., Slemenda, S.B., Pieniazek, N.J., 2002. Mole-
cular characterization of Babesia canis canis and Babesia canis
vogeli from naturally infected European dogs. Vet. Parasitol.
106, 285–292.
Camacho, A.T., Pallas, E., Gestal, J.J., Guitian, F.J., Olmeda, A.S.,
Goethert, H.K., Telford, S.R., 2001. Infection of dogs in North-
west Spain with a Babesia microti-like agent. Vet. Rec. 149,
552–555.
Carret, C., Walas, F., Carcy, B., Grande, N., Precigout, E., Moubri,
K., Schetters, T.P.M., Gorenflot, A., 1999. Babesia canis canis,
Babesia canis vogeli, Babesia canis rossi: differentiation of the
three subspecies by a restriction fragment length polymorphism
analysis on amplified small subunit ribosomal RNA genes. J.
Eukaryot. Microbiol. 46, 298–303.
Criado-Fornelio, A., Martinez-Marcos, A., Buling-Sarana, A., Barba-
Carretero, J.C., 2003. Molecular studies on Babesia, Theileria,
and Hepatozoon in Southern Europe. Part II. Phylogenetic ana-
lysis and evolutionary history. Vet. Parasitol. 114, 173–194.
Depoix, D., Carcy, B., Jumas-Bilak, E., Pages, M., Precigout, E.,
Schetters, T.P.M., Ravel, C., Gorenflot, A., 2002. Chromosome
number, genome size and polymorphism of European and South
African isolates of large Babesia parasites that infect dogs.
Parasitology 125, 313–321.
Duh, D., Tozon, N., Petrovec, M., Strasek, K., Avsic-Zupanc, T.,
2004. Canine babesiosis in Slovenia: molecular evidence of
Babesia canis canis and Babesia canis vogeli. Vet. Res. 35,
363–368.
Estrada-Pena, A., Bouattour, A., Camicas, J.L., Walter, A.R., 2004.
Tick of domestic animals in the mediterranean region: a guide to
identification of species. Int. Consort. Ticks Tick-Borne Dis.
124–127.
Foldvari, G., Hell, E., Farkas, R., 2005. Babesia canis canis in dogs
from Hungary: detection by PCR and sequencing. Vet. Parasitol.
127, 221–226.
Jefferies, R., Ryan, U.M., Muhlnickel, C.J., Irwin, P.J., 2003. Two
species of canine Babesia in Australia: detection and character-
ization by PCR. J. Parasitol. 89, 409–412.
Inokuma, H., Yoshizaki, Y., Matsumoto, K., Okuda, M., Onishi, T.,
Nakagome, K., Kosugi, R., Hirakawa, M., 2004. Molecular
survey of Babesia infection in dogs in Okinawa. Jpn. Vet.
Parasitol. 121, 341–346.
Kjemtrup, A.M., Kocan, A.A., Whitworth, L., Meinkoth, J., Bir-
kenheuer, A.J., Cummings, J., Boudreaux, M.K., Stockhan, S.L.,
Irizarry-Rovira, A., Conrad, P.A., 2000. There are at least three
genetically distinct small piroplasms from dogs. Int. J. Parasitol.
30, 1501–1505.
Kuttler, K.L., 1988. World-wide impact of babesiosis. In: Ristic, M.
(Ed.), Babesiosis of Domestic Animals and Man. CRC Press,
Boca Raton, FL, pp. 1–22.
Martin, A.R., Dunstan, R.H., Roberts, T.K., Brown, G.K., 2006.
Babesia canis vogeli: a novel PCR for its detection in dogs in
Australia. Vet. Parasitol. 112, 63–65.
Matjila, P.T., Penzhorn, B.L., Bekker, C.P.J., Nijhof, A.M., Jongejan,
F., 2004. Confirmation of occurrence of Babesia canis vogeli in
domestic dogs in South Africa. Vet. Parasitol. 122, 119–125.
Matjila, P.T., Nijhof, A.M., Taoufik, A., Houwers, D., Teske, E.,
Penzhorn, B.L., de Lange, T., Jongejan, F., 2005. Autochtho-
nous canine babesiosis in the Netherlands. Vet. Parasitol. 131,
23–29.
Olmeda, A.S., Armstrong, P.M., Rosenthal, B.M., Valladares, B.,
Del castillo, A., De Armas, F., Miguelez, M., Gonzales, A.,
Rodriguez, J.A., Spielman, A., Telford III, S.R., 1997. A sub-
tropical case of human babesiosis. Acta Tropica 37, 229–234.
Oyamada, M., Davoust, B., Boni, M., Dereure, J., Bucheton, B.,
Hammad, A., Itamoto, K., Okuda, M., Inokuma, H., 2005.
Detection of Babesia canis rossi, B. canis vogeli, and Hepato-
zoon canis in dogs in a village of eastern Sudan by using a
screening PCR and sequencing methodologies. Clin. Diagn.
Lab. Immunol. 12, 1343–1346.
Passos, L.M.F., Geiger, S.M., Ribeiro, M.F.B., Pfister, K., Zahler-
Rinder, M., 2005. First molecular detection of Babesia vogeli in
dogs from Brazil. Vet. Parasitol. 127, 81–85.
Patton, W.S., 1910. Rapport preliminaire sur un nouveau piro-
plasme, P. gibsoni nov. sp. trouve dans le chien de madras et
du chacal Canis aureus. Bull. Soc. Path. Exo. 3, 274–280.
Piana, G.P., Galli-Valerio, B., 1895. Su di un’infezione del cane con
parassiti endoglobulari nel sangue. Nota preventiva. Il Moderno
Zooiatro 6, 163–169.
Sanger, E., Nicklen, S., Coulson, A.R., 1977. DNA sequencing with
chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74,
5463–5467.
Schetters, T.P.M., Moubri, K., Precigout, E., Kleuskens, J., Scholtes,
N.C., Gorenflot, A., 1997. Different Babesia canis isolates,
different diseases. Parasitology 115, 485–493.
A. Gulanber et al. / Veterinary Parasitology 139 (2006) 224–230230
Schetters, T.P.M., 2005. Vaccination against canine babesiosis.
Trends Parasitol. 21, 179–184.
Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W:
improving the sensitivity of progressive multiple sequence
alignment through sequence weighting, positions-specific gap
penalties and weight matrix choice. Nucleic Acids Res. 22,
4673–4680.
Uilenberg, G., Franssen, F.F.J., Perie, M., Spanjer, A.A.M., 1989.
Three groups of Babesia canis distinguished and a proposal for
nomenclature. Vet. Q. 11, 33–40.
Zahler, M., Schein, E., Rinder, H., Gothe, R., 1998. Characteristic
genotypes discriminate between Babesia canis isolates of dif-
fering vector specificity and pathogenicity to dogs. Parasitol.
Res. 84, 544–548.
Zahler, M., Rinder, H., Zweygarth, E., Fukata, T., Maede, Y., Schein,
E., Gothe, R., 2000a. Babesia gibsoni of dogs from North America
and Asia belong to different species. Parasitology 120, 365–369.
Zahler, M., Rinder, H., Schein, E., Gothe, R., 2000b. Detection of a
new Babesia microti-like species in dogs. Vet. Parasitol. 89,
241–248.