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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 canis vogeli from Japan, B. canis canis from UK, and B.
canis rossi from Sudan. Other related Babesia spp. and
Babesia gibsoni and Babesia canis are well-known Theileria spp. were also analyzed and compared.
Babesia species in canine hosts (Yonamine et al., 1984).
B. canis has been classified into three subtypes, B. canis 2. Materials and methods
vogeli, B. canis canis, and B. canis rossi, according to
genetic data, vector specificity, and variations in 2.1. Piroplasm isolates
pathogenicity (Uilenberg et al., 1989; Zahler et al.,
1998; Carret et al., 1999). Moreover, an increased number B. gibsoni isolates obtained from two naturally
of genetically unique piroplasms have been identified, infected dogs from Okinawa (BgOki61 and BgOki79),
and the recognized geographic ranges of various canine Japan, and B. gibsoni isolates reported previously (Gen-
piroplasms appear to be expanding (Birkenheuer et al., Bank accession number AB083510; Japan1, AB083511;
1999; Conrad et al., 1991; Kjemtrup and Kocan et al., Japan2, AB083512; Korea1, and AB083513; Korea2)
2000; Zahler, Rinder, Zweygarth et al., 2000; Camacho were used in the present study (Yamasaki et al., 2002). B.
et al., 2001; Irizarry-Rovira et al., 2001; Kocan et al., canis vogeli isolates were obtained from two naturally
2001; Macintire et al., 2002; Birkenheuer et al., 2004). infected dogs from Okinawa (BcvOki64 and BcvOki76),
Since canine babesiosis occurs worldwide and causes Japan. B. canis canis isolates were obtained from two
hemolytic anemia and thrombocytopenia in dogs, it is naturally infected dogs, which were diagnosed at the
important to classify the species, subspecies, and School of Clinical Vet Science, University of Bristol, UK
genotype of the parasites to avoid confusion in the (Bcc295 and Bcc450). B. canis rossi isolates were
clinical setting (Birkenheuer et al., 1999). Recently, obtained from three naturally infected dogs from Sudan
molecular techniques including the polymerase chain (Bcr44, Bcr69, and Bcr76). The B. divergens isolate was
reaction (PCR) and sequencing have been used for the obtained from a naturally infected cow from the UK
epidemiological study and phylogenetic analysis of (BdivUK). The B. odocoilei isolate was obtained from a
piroplasms because of their sensitivity (Burkot et al., naturally infected white-tailed deer from the USA
2000; Kjemtrup and Thomford et al., 2000; Duh et al., (Bodo625-3). The B. ovis isolate was obtained from a
2001; Holman et al., 2002). In most of these studies, the naturally infected sheep from Turkey (BovTur). The
genes of 18S nuclear subunit ribosomal DNA (18S rDNA) USDA strain of B. caballi (BcabUSDA) and USDA strain
from many Babesia and Theileria species were analyzed of B. equi (BeqUSDA) were obtained from experimen-
and utilized (Kjemtrup and Kocan et al., 2000; Kjemtrup tally infected horses. B. microti strain Gray and B. microti
and Thomford et al., 2000; Zahler, Rinder, Zweygarth strain Gray/mo, kindly provided by Dr. Tsuneo
et al., 2000; Zahler, Rinder, Schein et al., 2000; Inokuma Kamiyama (Department of Veterinary Science, National
et al., 2003). The same genes have also been utilized for Institute of infectious diseases, Japan), were obtained
the detection and the differentiation of Babesia and from experimentally infected mice (Matsubara et al.,
Theileria species (Carret et al., 1999; Birkenheuer et al., 1993). The T. annulata (TanTur) isolate was obtained
1999; Altay et al., 2005; Aktas et al., 2005). Ideally, from a naturally infected cow in Turkey. The T. ovis
however, a phylogenetic analysis should be based on the (TovTur) isolate was obtained from a naturally infected
sequences of multiple genes in addition to 18S rDNA. sheep in Turkey. The T. cervi (Tcer621-5) isolate was
Previously (Yamasaki et al., 2002), we analyzed the obtained from a naturally infected white-tailed deer from
sequence of the heat shock protein 70 (hsp70) gene of B. the USA. The piroplasm isolates with the exception of B.
gibsoni and compared it with the corresponding gibsoni, B. canis vogeli, and B. microti were provided as
sequences from Babesia bovis, Babesia microti, extracted genomic DNAs from the infected animals. The
Theileria annulata, and Theileria sergenti renamed T. sequences of the hsp70 genes of B. bovis (AF107118), B.
orientalis. B. gibsoni is most closely related to B. bovis, rodhaini (AB103587), T. annulata (J04653), and T.
and has a closer genetic relationship with T. annulata sergenti, the latter renamed T. orientalis (D12692), were
and T. orientalis than with B. microti (Yamasaki et al., obtained from GenBank.
2002). Similar results were obtained in an analysis
based on the 18S rDNA of intraerythrocytic protozoan 2.2. DNA extraction, amplification, cloning, and
parasites (Kjemtrup and Thomford et al., 2000). sequencing
Therefore, an analysis of the hsp70 gene from
piroplasms might aid in the classification of piroplasms. Genomic DNA of piroplasms was extracted as
In the present study, we analyzed the hsp70 gene of B. 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 of the piroplasm isolates. The extracted genomic DNA
animal was lysed with 5 mM phosphate-buffer (PB; pH in reaction mixtures made according to the protocol
7.4) and centrifuged at 12,000  g for 10 min at 4 8C. supplied (ExTaq polymerase, Takara, Tokyo, Japan) was
After removal of the supernatant, genomic DNA was amplified for 35 cycles (94 8C, 1 min; 55 8C, 2 min;
extracted and used as a template for PCR. 72 8C, 3 min) in an AB-1820 thermocycler (ATTO,
The PCR primers used for the amplification of the Tokyo, Japan). In the B. gibsoni, B. canis canis, B.
entire or partial hsp70 gene of piroplasms were divergens, and B. ovis isolates, the PCR was performed
designed based on sequences conserved among the with four primer pairs; BabeF0-BabeR0, BabeF0-
hsp70 cDNAs of B. gibsoni, B. bovis, B. rodhaini, T. BabeR1, BabeF1-BabeR0, and BabeF1-BabeR1
annulata, and T. sergenti. These primers had a (Table 2). In the B. canis vogeli isolates, the PCR
degeneracy to allow for different bases. Various was performed with five primer pairs; BabeF0-BabeR4,
combinations of these primers were tested for the BabeF1-BabeR3, BabeF4-BabeR0, BabeF2-BabeR3,
amplification of the hsp70 gene from the genomic DNA 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; Rapid PCR Purification System (Marligen Biosciences
BabeF0-BabeR6, BabeF1-BabeR1, BabeF5-BabeR0, Inc., Maryland, USA) according to the directions
BabeF2-BabeR3, and BabeF1-BabeR2 (Table 2). Each supplied. The purified products in reaction mixtures
reaction product was checked by electrophoresis on a were amplified for 35 cycles (94 8C, 1 min; 60 8C,
1.5% agarose gel to confirm that it was a single product 1 min; 72 8C, 1 min). For those isolates, the nested PCR
and directly utilized for the sequencing analysis. The was performed with the multiple primer pairs listed in
primers used for the amplification were also utilized for Table 2. Each nested PCR product was checked by
the first sequencing analysis. The sequencing primers electrophoresis on a 1.5% agarose gel to confirm that it
used for the continued sequencing analysis were was a single product and utilized for the sequencing
designed based on the analyzed nucleotide sequence analysis.
(Tables 1 and 2, Fig. 1). For the B. canis rossi, B. The nucleotide sequences of the amplification
odocoilei, B. caballi, B. equi, T annulata, T. cervi and T. products were determined as described previously
ovis isolates, not enough primary PCR product was (Yamasaki et al., 2002). To compensate for the possible
obtained with the primer pair BabeF0-BabeR0 for direct misincorportation of nucleotides from the Taq poly-
sequencing and so the nested PCR method was used. merase, a minimum of three amplified products were
The products of the primary PCR were purified using a 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 = 97%) (Fig. 3a).
statistical support (Bootstrap value = 100%) (Fig. 3a). Moreover, B. microti and B. rodhaini formed
B. canis vogeli, B. canis canis, and B. canis rossi were group C with good statistical support (Bootstrap
particularly closely related even with poor statistical value = 100%), which was closely related to P.
support (Bootstap value = 77%). T. annulata, T. falciparum (Fig. 3a). A phylogenetic analysis based
orientalis, and T. cervi made up group B with good on the complete amino acid sequence of the hsp70
statistical support (Bootstrap value = 100%) (Fig. 3a). protein from piroplasms gave a similar result to that
B. equi was closely related to group A and B with good 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 Indeed, a phylogenetic analysis based on the hsp70
similar to each other. The hsp70 genes cloned from the gene showed that piroplasms could be classified into
extracted genomic DNA of the B. canis family were three groups with good statistical support, and canine
almost the same length as the gene from B. gibsoni. It babesial isolates made up one cluster in one group.
had been reported that the hsp70 gene of B. gibsoni has Additionally, the analyzed hsp70 genes of piroplasm
no intron (Yamasaki et al., 2002). Therefore, the present isolates would have no introns, indicating that they
results suggested that the hsp70 genes of the B. canis might also be heat inducible hsp70s and could be
family would have no introns either. The hsp70 family compared to each other. The results of the analysis
are divided into three groups based on expression based on the amino acid sequence of hsp70 were almost
patterns: those that are constitutively expressed (heat the same as those based on the sequence of the hsp70
shock cognate protein or hsc70) and those that are heat gene. Similar results have been obtained in an analysis
inducible (hsp70) or glucose-regulated (grp78) (Jie based on the 18S rDNA of intraerythrocytic protozoan
et al., 1996). The intronless state may be suggestive of parasites (Kjemtrup and Kocan et al., 2000). These
heat inducibility since inducible forms of hsp70 have results indicated that the nucleotide sequence and
been reported to lack introns (Gunther and Walter, amino acid sequence of hsp70 are helpful for the
1994). From these previous reports and the present classification of piroplasms, and suggested that B.
results, it was supposed that the hsp70 genes analyzed in gibsoni, B. canis canis, B. canis vogeli, and B. canis
the present study might be heat inducible hsp70s and rossi are very closely related and may have the same
might play the same important role within each parasite. ancestry. However, slightly different results of a
Therefore, it was considered that these hsp70 genes phylogenetic analysis based on the 18S rDNA sequence
from canine babesial isolates could be compared. The have also been reported (Zahler, Rinder, Zweygarth
predicted amino acid sequences of the hsp70 proteins of et al., 2000; Zahler, Rinder, Schein et al., 2000;
those Babesia species and subspecies were also very Birkenheuer et al., 2004), in which B. gibsoni and B.
similar to each other, with about 520 amino acids of the canis did not make one cluster, though they formed one
amino terminal side almost the same. Jie et al. suggested group with other Babesia species. In the present study,
that hsp70 proteins perform functions critical to the canine babesial isolates had made up one cluster in one
survival of B. microti parasites (Jie et al., 1996). The group with poor statistical support. Therefore, it is
hsp70 of protozoan parasites might play important roles possible that the canine babesial isolates might be
in their survival within the host (Van der Ploeg et al., found to form another cluster on further study.
1985; Lindquist, 1986; Shapira et al., 1988; Kaufmann, Moreover, several small Babesia species, which were
1990). For example, a temperature increase from 25 to closely related to B. microti, B. rodhaini, and B. equi,
37 8C induces a significant heat-shock response in have been isolated from dogs (Zahler, Rinder, Schein
certain leishmania promastigotes in vitro, resulting in et al., 2000). The new canine babesial isolates, from
their differentiation (Van der Ploeg et al., 1985; Shapira California, were in a distinct phylogenetic clade,
et al., 1988). Besides elevations in temperature, closely related to babesial isolates from wildlife and
parasites are exposed to many other stressful stimuli humans from the Western US according to an analysis
within the host, particularly those mediated by immune- based on the 18S rDNA gene (Kjemtrup and Kocan
defense mechanisms (Kaufmann, 1990). These reports et al., 2000). A novel large Babesia sp. that is a
indicated that hsp70 is an important functional protein genetically distinct piroplasm could infect the domestic
for protozoan parasites, the structure of which varies dog (Birkenheuer et al., 2004). To clarify the
little among closely related species. Therefore, it is classification of those novel canine babesial isolates,
supposed that the region of hsp70 well conserved more phylogenetic analyses based on multiple genes,
among canine babesial isolates is of functional such as hsp70, will be needed. Phylogenetic analyses of
importance and so changes little. Because hsp70 may the hsp70 gene sequence should aid in the classification
be an important functional protein, the degree of of Babesia and Theileria species.
difference in its structure might reflect the distance
across species of protozoan parasites. Moreover, the Acknowledgements
present study showed that the nucleotide sequences of
the hsp70 gene from canine babesial isolates had more This work was supported in part by a Grant of the
variety than those of the 18S rDNA gene, suggesting 21st Century COE Program, Program of Excellence
that the former would aid in the classification of for Zoonosis Control, Ministry of Education, Science,
Babesia and Theileria species. Sports and Culture of Japan.
M. Yamasaki et al. / Veterinary Parasitology 145 (2007) 217227 227

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