Veterinary Parasitology 145 (2007) 217227

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Comparison and phylogenetic analysis of the heat shock protein

70 gene of Babesia parasites from dogs
Masahiro Yamasaki a,*, Hisashi Inokuma b, Chihiro Sugimoto c, Susan E. Shaw d,
Munir Aktas e, Michael J. Yabsley f, Osamu Yamato g, Yoshimitsu Maede a
a
Laboratory of Internal Medicine, Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine,
Hokkaido University, Sapporo 060-0818, Japan
b
Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan
c
Department of Collaboration and Education, Research Center for Zoonosis Control, Hokkaido University, Sapporo 060-0818, Japan
d
Acarus Laboratory, University of Bristol, School of Clinical Vet Science, Churchill Building, Langford, Bristol BS40 5DU, UK
e
Department of Parasitology, Faculty of Veterinary Medicine, University of Firat, 23119 Elazig, Turkey
f
Southeastern Cooperative Wildlife Disease Study, College of Veterinary Medicine, University of Georgia,
Athens, GA 30602, USA
g
Laboratory of Clinical Pathology, Department of Veterinary Clinical Sciences, Faculty of Agriculture,
Kagoshima University, 1-21-24 Kohrimoto, Kagoshima 890-0065, Japan
Received 15 July 2006; received in revised form 28 December 2006; accepted 2 January 2007

Abstract
The heat shock protein 70 (hsp70) genes of Babesia gibsoni, B. canis canis, B. canis vogeli, and B. canis rossi isolated from
infected dogs were cloned by polymerase chain reaction (PCR) and sequenced. In the nucleotide sequence and the predicted amino
acid sequence of the gene, the parasites were very similar to each other. The nucleotide sequences of the hsp70 gene had more
variety than those of 18S nuclear subunit ribosomal DNA (18S rDNA). A phylogenetic analysis of these sequences and comparisons
with sequences from other Babesia and Theileria species revealed that all canine babesial isolates analyzed in the present study were
closely related to each other and formed one cluster. Additionally, a phylogenetic analysis of Babesia and Theileria species showed
that these parasites could be divided into three groups: group A including canine babesial isolates, B. divergens, B. odocoilei, B.
bovis, B. caballi, and B. ovis; group B including Theileria annulata, T. orientalis, and T. cervi; and group C including B. microti and
B. rodhaini. These results suggested that a phylogenetic analysis of the hsp70 gene sequence might be helpful in classifying Babesia
and Theileria species, and that canine babesial isolates might be closely related to each other, indicating their evolution from the
same ancestry.
# 2007 Elsevier B.V. All rights reserved.

Keywords: Babesia gibsoni; Babesia canis vogeli; Babesia canis canis; Babesia canis rossi; Heat shock protein 70; Phylogenetic analysis

Abbreviations: PCR, polymerase chain reaction; hsp70, heat shock protein 70

Note: Nucleotide sequence data reported in this paper are available in the GenBankTM, EMBL and DDBJ database under the accession numbers:
AB248730 (BgOki61), AB248731 (BgOki79), AB248732 (BcvOki64), AB248733 (BcvOki76), AB248734 (Bcc295), AB248735 (Bcc450),
AB248736 (Bcr44), AB248737 (Bcr69), AB248738 (Bcr76), AB248739 (BdivUK), AB248740 (Bodo625-3), AB248741 (BovTur), AB248742
(BcabUSDA), AB248743 (BeqUSDA), AB248744 (B. microti Gray strain), AB248745 (B. microti Gray/mo strain), AB248746 (TanTur),
AB248747 (TovTur), AB248748 (Tcer621-5).
* Corresponding author. Tel.: +81 11 706 5223; fax: +81 11 709 7296.
E-mail address: masayama@vetmed.hokudai.ac.jp (M. Yamasaki).

0304-4017/$ see front matter # 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetpar.2007.01.003
218 M. Yamasaki et al. / Veterinary Parasitology 145 (2007) 217227
1. Introduction
Babesia gibsoni and Babesia canis are well-known
Babesia species in canine hosts (Yonamine et al., 1984).
B. canis has been classified into three subtypes, B. canis
vogeli, B. canis canis, and B. canis rossi, according to
genetic data, vector specificity, and variations in
pathogenicity (Uilenberg et al., 1989; Zahler et al.,
1998; Carret et al., 1999). Moreover, an increased number
of genetically unique piroplasms have been identified,
and the recognized geographic ranges of various canine
piroplasms appear to be expanding (Birkenheuer et al.,
1999; Conrad et al., 1991; Kjemtrup and Kocan et al.,
2000; Zahler, Rinder, Zweygarth et al., 2000; Camacho
et al., 2001; Irizarry-Rovira et al., 2001; Kocan et al.,
2001; Macintire et al., 2002; Birkenheuer et al., 2004).
Since canine babesiosis occurs worldwide and causes
hemolytic anemia and thrombocytopenia in dogs, it is
important to classify the species, subspecies, and
genotype of the parasites to avoid confusion in the
clinical setting (Birkenheuer et al., 1999). Recently,
molecular techniques including the polymerase chain
reaction (PCR) and sequencing have been used for the
epidemiological study and phylogenetic analysis of
piroplasms because of their sensitivity (Burkot et al.,
2000; Kjemtrup and Thomford et al., 2000; Duh et al.,
2001; Holman et al., 2002). In most of these studies, the
genes of 18S nuclear subunit ribosomal DNA (18S rDNA)
from many Babesia and Theileria species were analyzed
and utilized (Kjemtrup and Kocan et al., 2000; Kjemtrup
and Thomford et al., 2000; Zahler, Rinder, Zweygarth
et al., 2000; Zahler, Rinder, Schein et al., 2000; Inokuma
et al., 2003). The same genes have also been utilized for
the detection and the differentiation of Babesia and
Theileria species (Carret et al., 1999; Birkenheuer et al.,
1999; Altay et al., 2005; Aktas et al., 2005). Ideally,
however, a phylogenetic analysis should be based on the
sequences of multiple genes in addition to 18S rDNA.
Previously (Yamasaki et al., 2002), we analyzed the
sequence of the heat shock protein 70 (hsp70) gene of B.
gibsoni and compared it with the corresponding
sequences from Babesia bovis, Babesia microti,
Theileria annulata, and Theileria sergenti renamed T.
orientalis. B. gibsoni is most closely related to B. bovis,
and has a closer genetic relationship with T. annulata
and T. orientalis than with B. microti (Yamasaki et al.,
2002). Similar results were obtained in an analysis
based on the 18S rDNA of intraerythrocytic protozoan
parasites (Kjemtrup and Thomford et al., 2000).
Therefore, an analysis of the hsp70 gene from
piroplasms might aid in the classification of piroplasms.
In the present study, we analyzed the hsp70 gene of B.
canis vogeli from Japan, B. canis canis from UK, and B.
canis rossi from Sudan. Other related Babesia spp. and
Theileria spp. were also analyzed and compared.
2. Materials and methods
2.1. Piroplasm isolates
B. gibsoni isolates obtained from two naturally
infected dogs from Okinawa (BgOki61 and BgOki79),
Japan, and B. gibsoni isolates reported previously (Gen-
Bank accession number AB083510; Japan1, AB083511;
Japan2, AB083512; Korea1, and AB083513; Korea2)
were used in the present study (Yamasaki et al., 2002). B.
canis vogeli isolates were obtained from two naturally
infected dogs from Okinawa (BcvOki64 and BcvOki76),
Japan. B. canis canis isolates were obtained from two
naturally infected dogs, which were diagnosed at the
School of Clinical Vet Science, University of Bristol, UK
(Bcc295 and Bcc450). B. canis rossi isolates were
obtained from three naturally infected dogs from Sudan
(Bcr44, Bcr69, and Bcr76). The B. divergens isolate was
obtained from a naturally infected cow from the UK
(BdivUK). The B. odocoilei isolate was obtained from a
naturally infected white-tailed deer from the USA
(Bodo625-3). The B. ovis isolate was obtained from a
naturally infected sheep from Turkey (BovTur). The
USDA strain of B. caballi (BcabUSDA) and USDA strain
of B. equi (BeqUSDA) were obtained from experimen-
tally infected horses. B. microti strain Gray and B. microti
strain Gray/mo, kindly provided by Dr. Tsuneo
Kamiyama (Department of Veterinary Science, National
Institute of infectious diseases, Japan), were obtained
from experimentally infected mice (Matsubara et al.,
1993). The T. annulata (TanTur) isolate was obtained
from a naturally infected cow in Turkey. The T. ovis
(TovTur) isolate was obtained from a naturally infected
sheep in Turkey. The T. cervi (Tcer621-5) isolate was
obtained from a naturally infected white-tailed deer from
the USA. The piroplasm isolates with the exception of B.
gibsoni, B. canis vogeli, and B. microti were provided as
extracted genomic DNAs from the infected animals. The
sequences of the hsp70 genes of B. bovis (AF107118), B.
rodhaini (AB103587), T. annulata (J04653), and T.
sergenti, the latter renamed T. orientalis (D12692), were
obtained from GenBank.
2.2. DNA extraction, amplification, cloning, and
sequencing
Genomic DNA of piroplasms was extracted as
described previously (Yamasaki et al., 2002). A 50-ml
 M. Yamasaki et al. / Veterinary Parasitology 145 (2007) 217227 219

volume of packed erythrocytes from each infected
animal was lysed with 5 mM phosphate-buffer (PB; pH
7.4) and centrifuged at 12,000  g for 10 min at 4 8C.
After removal of the supernatant, genomic DNA was
extracted and used as a template for PCR.
The PCR primers used for the amplification of the
entire or partial hsp70 gene of piroplasms were
designed based on sequences conserved among the
hsp70 cDNAs of B. gibsoni, B. bovis, B. rodhaini, T.
annulata, and T. sergenti. These primers had a
degeneracy to allow for different bases. Various
combinations of these primers were tested for the
amplification of the hsp70 gene from the genomic DNA
of the piroplasm isolates. The extracted genomic DNA
in reaction mixtures made according to the protocol
supplied (ExTaq polymerase, Takara, Tokyo, Japan) was
amplified for 35 cycles (94 8C, 1 min; 55 8C, 2 min;
72 8C, 3 min) in an AB-1820 thermocycler (ATTO,
Tokyo, Japan). In the B. gibsoni, B. canis canis, B.
divergens, and B. ovis isolates, the PCR was performed
with four primer pairs; BabeF0-BabeR0, BabeF0-
BabeR1, BabeF1-BabeR0, and BabeF1-BabeR1
(Table 2). In the B. canis vogeli isolates, the PCR
was performed with five primer pairs; BabeF0-BabeR4,
BabeF1-BabeR3, BabeF4-BabeR0, BabeF2-BabeR3,
and BabeF1-BabeR1 (Table 2). In the B. microti

Table 1
Oligonucleotides for the analysis of the hsp70 gene of piroplasms
Name Sequences Use
0 0
BabeF0 5 -atgdcwgshccmgctatyggwattgacttggg-3 Amplification/sequencing
BabeF1 50 -awgatatgaarcactggcca-30 Amplification/sequencing
BabeF2 50 -atgymtgstccagctattgg-30 Amplification/sequencing
BabeF3 50 -atcttcgaagtcaaggccac-30 Amplification/sequencing
BabeF4 50 -tcaaggacttcttcaacgga-30 Amplification/sequencing
BabeF5 50 -agaacgttctatgacatgtg-30 Amplification/sequencing
BabeF6 50 -gatcgaggttaccttcgata-30 Sequencing
BabeF7 50 -tccacctcacyggtattgcc-30 Sequencing
BabeF8 50 -caaytsgagaactactgcta-30 Sequencing
BabeF9 50 -aagtgcatcgaatctaagca-30 Sequencing
BabeF10 50 -gacacccatctgggaggtga-30 Sequencing
BabeF11 50 -tccaccccgaggaaatttca-30 Sequencing
BabeR0a 50 -cwtgtghttagtcaacytcctcwac-30 Amplification/sequencing
BabeR1a 50 -cccttgtcgttggtratrgt-30 Amplification/sequencing
BabeR2a 50 -tcaacytcctcwacagtggg-30 Amplification/sequencing
BabeR3a 50 -magmacvgtcatgacaccac-30 Amplification/sequencing
BabeR4a 50 -cacacatctcctcgaaacga-30 Amplification/sequencing
BabeR5a 50 -ttagtcaacttcctctacagtgg-30 Amplification/sequencing
BabeR6a 50 -accagcgtccttagtggcct-30 Amplification/sequencing
BabeR7a 50 -ggatggtcagcctggacggc-30 Sequencing
BabeR8a 50 -agttgtcaaagtcttcacca-30 Sequencing
BabeR9a 50 -ccacctccaagrtcgaagat-30 Sequencing
BabeR10a 50 -gggtggaakgtcttcttctg-30 Sequencing
BabeR11a 50 -ccaccacctagatcaaaaat-30 Sequencing
BabeR12a 50 -gcatggaatgtcttcttttg-30 Sequencing
BabeR13a 50 -aaggtaaagctcggcaattt-30 Sequencing
BabeR14a 50 -tgaccttgaatggccagtgc-30 Sequencing
TheiF0 50 -cwgcmgtccargctgctatc-30 Amplification/sequencing
TheiF1 50 -tgatacaggtgtttgaaggg-30 Amplification/sequencing
TheiF2 50 -aagayccargamttgctcct-30 Sequencing
TheiF3 50 -asrtyaccatcaccaacgac-30 Sequencing
TheiF4 50 -gtgagaagaagacgttccat-30 Sequencing
TheiF5 50 -tgacgaaggataataacctg-30 Sequencing
TheiF6 50 -cacaagctggagaattattg-30 Sequencing
TheiR0a 50 -cckagcargttgtwgtcctt-30 Sequencing
TheiR1a 50 -ytccttkccgytgaagaagt-30 Sequencing
TheiR2a 50 -gtwccaccwcccaartcgaa-30 Sequencing
TheiR3a 50 -attgtaggctttccacttgg-30 Sequencing
a
Antisense primers.
220 M. Yamasaki et al. / Veterinary Parasitology 145 (2007) 217227

Table 2
Fragments for the analysis of the hsp70 gene of piroplasms, and oligonucleotides for the sequencing
Species First fragmentsa Second fragmentsb Sequencingc
B. gibsoni BabeF0-BabeR0 BabeF1, BabeF3
BabeF0-BabeR1 BabeF4, BabeF6
BabeF1-BabeR0 BabeR14
BabeF1-BabeR1
B. canis vogeli BabeF0-BabeR4 BabeF4, BabeF7
BabeF1-BabeR3 BabeF8, BabeR2
BabeF4-BabeR0 BabeR3, BabeR4
BabeF2-BabeR3 BabeR9, BabeR10
BabeF1-BabeR1
B. canis canis BabeF0-BabeR0 BabeF4, BabeF8
BabeF0-BabeR1 BabeR3, BabeR4
BabeF1-BabeR0 BabeR9
BabeF1-BabeR1
B. canis rossi BabeF0-BabeR0 BabeF2-BabeR2 BabeF1, BabeF4
BabeF1-BabeR3 BabeF6, BabeF10
BabeR1, BabeR13
B. divergens BabeF0-BabeR0 BabeF4, BabeF8
BabeF0-BabeR1 BabeR3, BabeR4
BabeF1-BabeR0 BabeR9
BabeF1-BabeR1
B. odocoilei BabeF0-BabeR0 BabeF2-BabeR4 BabeF4, BabeF8
BabeF3-BabeR3 BabeF11, BabeR2
BabeF4-BabeR2 BabeR3, BabeR8
BabeF1-BabeR3 BabeR14
B. ovis BabeF0-BabeR0 BabeF7, BabeR1
BabeF0-BabeR1 BabeR4, BabeR7
BabeF1-BabeR0 BabeR9, BabeR10
BabeF1-BabeR1
B. caballi BabeF0-BabeR0 BabeF2-BabeR4 BabeF2, BabeF3
BabeF3-BabeR3 BabeF4, BabeF8
BabeF4-BabeR2 BabeR2, BabeR3
BabeF3-BabeR1 BabeR4, BabeR9
BabeR10
B. equi BabeF0-BabeR0 BabeF2-BabeR3 BabeF2, BabeF3
BabeF4-BabeR2 BabeF4, BabeF9
BabeF1-BabeR3 BabeR2, BabeR3
BabeR4, BabeR11
BabeR12
B. microti BabeF0-BabeR6 BabeF1, BabeF5
BabeF1-BabeR1 BabeR1, BabeR6
BabeF5-BabeR0
BabeF2-BabeR3
BabeF1-BabeR2
T. annulata BabeF0-BabeR0 BabeF2-BabeR1 BabeR1, TheiF2
TheiF0-BabeR2 TheiF3, TheiR0
BabeF1-BabeR3 TheiR1, TheiR2
T. cervi BabeF0-BabeR0 BabeF2-BabeR1 BabeR1, BabeR9
TheiF1-BabeR5 BabeR14, TheiF1
BabeF1-BabeR3 TheiF4, TheiF5
TheiF6, TheiR1
TheiR3
 M. Yamasaki et al. / Veterinary Parasitology 145 (2007) 217227 221
Table 2 (Continued )
Species First fragments a Second fragments b Sequencing c
T. ovis BabeF0-BabeR0 BabeF2-BabeR1 BabeR1, TheiF2
TheiF0-BabeR2 TheiF3, TheiR0
BabeF3-BabeR1 TheiR1, TheiR2
a
Primer pair(s) for the primary PCR.
b
Primer pair(s) for the nested PCR.
c
Primers used for the sequencing analysis.

isolates, the PCR was performed with five primer pairs;
BabeF0-BabeR6, BabeF1-BabeR1, BabeF5-BabeR0,
BabeF2-BabeR3, and BabeF1-BabeR2 (Table 2). Each
reaction product was checked by electrophoresis on a
1.5% agarose gel to confirm that it was a single product
and directly utilized for the sequencing analysis. The
primers used for the amplification were also utilized for
the first sequencing analysis. The sequencing primers
used for the continued sequencing analysis were
designed based on the analyzed nucleotide sequence
(Tables 1 and 2, Fig. 1). For the B. canis rossi, B.
odocoilei, B. caballi, B. equi, T annulata, T. cervi and T.
ovis isolates, not enough primary PCR product was
obtained with the primer pair BabeF0-BabeR0 for direct
sequencing and so the nested PCR method was used.
The products of the primary PCR were purified using a
Rapid PCR Purification System (Marligen Biosciences
Inc., Maryland, USA) according to the directions
supplied. The purified products in reaction mixtures
were amplified for 35 cycles (94 8C, 1 min; 60 8C,
1 min; 72 8C, 1 min). For those isolates, the nested PCR
was performed with the multiple primer pairs listed in
Table 2. Each nested PCR product was checked by
electrophoresis on a 1.5% agarose gel to confirm that it
was a single product and utilized for the sequencing
analysis.
The nucleotide sequences of the amplification
products were determined as described previously
(Yamasaki et al., 2002). To compensate for the possible
misincorportation of nucleotides from the Taq poly-
merase, a minimum of three amplified products were
sequenced directly. The comparison and analysis of the

Fig. 1. A map of the annealing location of each oligonucleotide for the analysis of the hsp70 gene from piroplasms. Arrowheads indicate annealing
locations of oligonucleotides on the heat shock protein 70 (hsp70) gene. The right-pointing arrowheads indicate sense primers, and the left-pointing
arrowheads, antisense primers. Numbers on the line indicate nucleotide numbers of the hsp70 gene of piroplasms.
222 M. Yamasaki et al. / Veterinary Parasitology 145 (2007) 217227

nucleotide sequences and the amino acid sequences Table 4

were performed using GENETYX-MAC ver. 11.2 Comparison of 18SrDNA genes of canine babesial isolates
(Genetyx Co., Tokyo, Japan). In the present study, a Homology (%)
number of PCR products amplified with various primer B. canis B. canis B. canis
pairs were analyzed for each isolate. The nucleotide vogeli canis rossi
sequences of these PCR products overlapped and B. gibsoni 94.4 94.7 94.9
matched perfectly. According to the nucleotide sequen- B. canis vogeli 97.6 94.9
cing analysis, the hsp70 genes from all piroplasm B. canis canis a
95.4
a a
isolates were about 1,940 bp long, and encoded a B. canis rossi
peptide of about 650 amino acids. The 18S rDNA genes a
Same value as in the corresponding column.
of B. gibsoni (Asia 1, AF175300), B. canis vogeli (USA,
AY371198), B. canis canis (AY072926), and B. canis
rossi (Dog-44, DQ111760) were also compared with Table 5
Comparison of the predicted amino acid sequences of hsp70 of canine
each other to observe any variety. A phylogenetic tree
babesial isolates
was inferred using ClustalX ver. 1.81 by the neighbor-
joining method (Thompson et al., 1997). Homology (%)
B. canis B. canis B. canis
3. Results vogeli canis rossi
B. gibsoni 96.3 96.3 96.4
3.1. Comparison of hsp70 genes from canine B. canis vogeli 99.2 98.0
a
babesial isolates B. canis canis 97.8
a a
B. canis rossi
a
In the present study, the hsp70 genes from B. gibsoni, Same value as in the corresponding column.
B. canis vogeli, and B. canis canis, were amplified by
primary PCR. However, as the hsp70 gene from B. canis
rossi was not amplified enough by primary PCR, nested The predicted amino acid sequence of hsp70 from B.
PCR procedures were utilized for the sequencing gibsoni was also compared with that of B. canis
analysis. According to the sequence analysis of the vogeli, B. canis canis, and B. canis rossi, and found to
PCR products from B. canis vogeli, B. canis canis, and show 96.3, 96.3, and 96.4% identity, respectively
B. canis rossi, the hsp70 genes from the genomic DNA (Table 5). Interestingly, three amino acids of the
of canine babesial isolates were similar to each other amino terminal side and 120 amino acids of the
(Table 3) and would have no introns (data not shown). carboxyl terminal side of the predicted amino acid
The nucleotide sequence of the gene from B. gibsoni sequences were characteristic for each species, the
was compared with that of B. canis vogeli, B. canis remaining parts being almost the same among canine
canis, and B. canis rossi, and found to have 86.6, 86.2, babesial isolates (Fig. 2).
and 86.5% identity, respectively (Table 3). Additionally,
the reported 18S rDNA gene from B. gibsoni (Asia1) 3.2. Phylogenetic analysis of hsp70 of piroplasms
was homologous with that of B. canis vogeli (USA), B.
canis canis, and B. canis rossi (Dog-44), exhibiting In addition to the sequences from the canine babesial
94.4, 94.7, and 94.9% identity, respectively (Table 4). isolates, the nucleotide and predicted amino acid
sequences of hsp70 from B. divergens, B. odocoilei, B
caballi, B. equi, B. ovis, B. microti, T. annulata, T. cervi,
Table 3
and T. ovis, which were cloned and analyzed in the
Comparison of hsp70 genes of canine babesial isolates
present study, were included in the phylogenetic
Homology (%) analysis (Fig. 3). The hsp70 genes from the genomic
B. canis B. canis B. canis DNA of all piroplasm isolates would have no intron the
vogeli canis rossi same as canine babesial isolates (data not shown). The
B. gibsoni 86.6 86.2 86.5 nucleotide and amino acid sequences of hsp70 for B.
B. canis vogeli 95.7 89.1 bovis, B. rodhaini, T. annulata, and T. orientalis from
a
B. canis canis 89.1 the GenBank database were also included. To estimate
a a
B. canis rossi the genetic distance from other species, the corre-
a
Same value as in the corresponding column. sponding hsp70 gene and amino acid sequence of
 M. Yamasaki et al. / Veterinary Parasitology 145 (2007) 217227 223

Fig. 2. Alignment of the predicted amino acid sequence of heat shock protein 70 from Babesia gibsoni, Babesia canis vogeli, Babesia canis canis,
and Babesia canis rossi. Identical residues are denoted by periods (.), while additions and gaps in the sequences are indicated by dashes (); Amino
acids, which are conserved among canine babesial isolates, are marked by asterisks (*).

Plasmodium falciparum (M19753) was also included piroplasms showed that canine babesial isolates were
in the analysis. A phylogenetic analysis based on the closely related to each other even with poor statistical
entire coding region of the hsp70 gene and complete support (Bootstrap value = 74%) and formed one
amino acid sequence of the hsp70 protein from cluster in group A, which includes B. bovis, B. ovis,
224 M. Yamasaki et al. / Veterinary Parasitology 145 (2007) 217227

B. caballi, B. divergens, and B. odocoilei, with good
statistical support (Bootstrap value = 100%) (Fig. 3a).
B. canis vogeli, B. canis canis, and B. canis rossi were
particularly closely related even with poor statistical
support (Bootstap value = 77%). T. annulata, T.
orientalis, and T. cervi made up group B with good
statistical support (Bootstrap value = 100%) (Fig. 3a).
B. equi was closely related to group A and B with good
statistical support (Bootstrap value = 97%) (Fig. 3a).
Moreover, B. microti and B. rodhaini formed
group C with good statistical support (Bootstrap
value = 100%), which was closely related to P.
falciparum (Fig. 3a). A phylogenetic analysis based
on the complete amino acid sequence of the hsp70
protein from piroplasms gave a similar result to that
based on the nucleotide sequence of the hsp70 gene

Fig. 3. Phylogenetic tree based on the heat shock protein 70 (hsp70) from piroplasms. The dendrogram was constructed with the neighbor-joining
method. (a) Phylogenetic relationship of piroplasms based on the full coding region of hsp70 genes. The numbers at the nodes indicate bootstrap
support from 1000 replications. The lengths of lines are proportional to the bootstrap value. The scale bar represents a 10% bootstrap value. (b)
Phylogenetic relationship of piroplasms based on the full amino acid sequences of hsp70. The numbers above branches indicate the rate of nucleotide
substitution per site. The lengths of the lines are proportional to the number of base changes. The scale bar represents a rate of 10 nucleotide
substitutions per 100 bases.
 M. Yamasaki et al. / Veterinary Parasitology 145 (2007) 217227 225

Fig. 3. (Continued ).

(Fig. 3b). T. ovis was closely related to P. falciparum 4. Discussion

based on the analysis of amino acid sequences (Fig. 3b),
while it was closely related to group A with good In the present study, the hsp70 genes from B. gibsoni,
statistical support (Bootstrap value = 100%) based on B. canis vogeli, B. canis canis, and B. canis rossi were
the analysis of nucleotide sequences (Fig. 3a). cloned and analyzed. The nucleotide sequences of this
226 M. Yamasaki et al. / Veterinary Parasitology 145 (2007) 217227
gene from B. gibsoni and three B. canis subspecies were
similar to each other. The hsp70 genes cloned from the
extracted genomic DNA of the B. canis family were
almost the same length as the gene from B. gibsoni. It
had been reported that the hsp70 gene of B. gibsoni has
no intron (Yamasaki et al., 2002). Therefore, the present
results suggested that the hsp70 genes of the B. canis
family would have no introns either. The hsp70 family
are divided into three groups based on expression
patterns: those that are constitutively expressed (heat
shock cognate protein or hsc70) and those that are heat
inducible (hsp70) or glucose-regulated (grp78) (Jie
et al., 1996). The intronless state may be suggestive of
heat inducibility since inducible forms of hsp70 have
been reported to lack introns (Gunther and Walter,
1994). From these previous reports and the present
results, it was supposed that the hsp70 genes analyzed in
the present study might be heat inducible hsp70s and
might play the same important role within each parasite.
Therefore, it was considered that these hsp70 genes
from canine babesial isolates could be compared. The
predicted amino acid sequences of the hsp70 proteins of
those Babesia species and subspecies were also very
similar to each other, with about 520 amino acids of the
amino terminal side almost the same. Jie et al. suggested
that hsp70 proteins perform functions critical to the
survival of B. microti parasites (Jie et al., 1996). The
hsp70 of protozoan parasites might play important roles
in their survival within the host (Van der Ploeg et al.,
1985; Lindquist, 1986; Shapira et al., 1988; Kaufmann,
1990). For example, a temperature increase from 25 to
37 8C induces a significant heat-shock response in
certain leishmania promastigotes in vitro, resulting in
their differentiation (Van der Ploeg et al., 1985; Shapira
et al., 1988). Besides elevations in temperature,
parasites are exposed to many other stressful stimuli
within the host, particularly those mediated by immune-
defense mechanisms (Kaufmann, 1990). These reports
indicated that hsp70 is an important functional protein
for protozoan parasites, the structure of which varies
little among closely related species. Therefore, it is
supposed that the region of hsp70 well conserved
among canine babesial isolates is of functional
importance and so changes little. Because hsp70 may
be an important functional protein, the degree of
difference in its structure might reflect the distance
across species of protozoan parasites. Moreover, the
present study showed that the nucleotide sequences of
the hsp70 gene from canine babesial isolates had more
variety than those of the 18S rDNA gene, suggesting
that the former would aid in the classification of
Babesia and Theileria species.
Indeed, a phylogenetic analysis based on the hsp70
gene showed that piroplasms could be classified into
three groups with good statistical support, and canine
babesial isolates made up one cluster in one group.
Additionally, the analyzed hsp70 genes of piroplasm
isolates would have no introns, indicating that they
might also be heat inducible hsp70s and could be
compared to each other. The results of the analysis
based on the amino acid sequence of hsp70 were almost
the same as those based on the sequence of the hsp70
gene. Similar results have been obtained in an analysis
based on the 18S rDNA of intraerythrocytic protozoan
parasites (Kjemtrup and Kocan et al., 2000). These
results indicated that the nucleotide sequence and
amino acid sequence of hsp70 are helpful for the
classification of piroplasms, and suggested that B.
gibsoni, B. canis canis, B. canis vogeli, and B. canis
rossi are very closely related and may have the same
ancestry. However, slightly different results of a
phylogenetic analysis based on the 18S rDNA sequence
have also been reported (Zahler, Rinder, Zweygarth
et al., 2000; Zahler, Rinder, Schein et al., 2000;
Birkenheuer et al., 2004), in which B. gibsoni and B.
canis did not make one cluster, though they formed one
group with other Babesia species. In the present study,
canine babesial isolates had made up one cluster in one
group with poor statistical support. Therefore, it is
possible that the canine babesial isolates might be
found to form another cluster on further study.
Moreover, several small Babesia species, which were
closely related to B. microti, B. rodhaini, and B. equi,
have been isolated from dogs (Zahler, Rinder, Schein
et al., 2000). The new canine babesial isolates, from
California, were in a distinct phylogenetic clade,
closely related to babesial isolates from wildlife and
humans from the Western US according to an analysis
based on the 18S rDNA gene (Kjemtrup and Kocan
et al., 2000). A novel large Babesia sp. that is a
genetically distinct piroplasm could infect the domestic
dog (Birkenheuer et al., 2004). To clarify the
classification of those novel canine babesial isolates,
more phylogenetic analyses based on multiple genes,
such as hsp70, will be needed. Phylogenetic analyses of
the hsp70 gene sequence should aid in the classification
of Babesia and Theileria species.
Acknowledgements
This work was supported in part by a Grant of the
21st Century COE Program, Program of Excellence
for Zoonosis Control, Ministry of Education, Science,
Sports and Culture of Japan.
 M. Yamasaki et al. / Veterinary Parasitology 145 (2007) 217227
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