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Babesia species are intra-erythrocytic apicomplexan parasites. They are transmitted by ticks and parasitize a wide range of vertebrate hosts. Babesia gibsoni causes canine babesiosis, which induces remittent fever, progressive anemia, thrombocytopenia, marked splenomegaly and hepatomegaly, and in some cases, the death of infected animals.1,2 Infection with B. gibsoni have been identified worldwide, and it is now recognized as a serious emergent disease in small animal medicine.3 Several chemotherapeutics, including diminazene aceturate,4 pentamidine isethionate,2 phenamidine isethionate,5 and clindamycin,6 have been used for treatment of B. gibsoni infections. However, these chemotherapeutics fail to eliminate B. gibsoni from host dogs completely.3,6 Therefore, an effective therapy is urgently needed.
Atovaquone is a novel antiprotozoal compound that has broad-spectrum activity against human protozoan pathogens, including Plasmodium spp., Toxoplasma gondii,7 and Babesia spp.8–11 This compound is an analog of ubiquinone, and its mechanism of action is by inhibition of mitochondrial electron transport.7,12 However, recurrence of disease and decreased sensitivity of protozoa to therapy have been reported when atovaquone alone was used. Mutation of the cytochrome b (cytb) gene, which is located in the mitochondrial genome, has been described in atovaquone-resistant isolates of Plasmodium spp.,13,14 T. gondii,15 and Pneumocystis carinii.16
A combination of atovaquone and azithromycin was reported to be effective for treatment of dogs that were naturally infected with B. gibsoni.17 In our previous study, we described the therapeutic efficacy of atovaquone against B. gibsoni using experimentally infected dogs, and suggested that the atovaquone allows recrudescence of parasites with decreased susceptibility to this drug.18 However, the mechanisms responsible have not been investigated. Genetic changes associated with decreased sensitivity of B. gibsoni to atovaquone may be valuable as markers for clinical application and for selection of drugs for use in combination with atovaquone. In this study, we identified a nucleotide coding sequence for B. gibsoni cytb, which might be the atovaquone target. Subsequently, the sequence of the cytb gene from atovaquone-treated animals with recurring parasitemia was determined and compared with that of the pretreatment parasite.
The Animal Care and Ethics Committee of Kitasato University approved the use of the animals in this study. The original parasite used in this study was isolated from a naturally infected Tosa dog in Aomori Prefecture, Japan, and was identified as an Asian genotype.19,20 This parasite was maintained in our laboratory by passage through a beagle (dog A) that was not exposed to drug treatment. Whole blood samples were collected from this dog, and EDTA was used as anticoagulant. Blood samples that had been stored in our laboratory were used to compare the nucleotide sequence of B. gibsoni cytb before and after atovaquone treatment. The blood samples were collected during our previous study from three experimentally infected dogs (B, C, and D) before atovaquone treatment and during the recurrence of B. gibsoni infection after atovaquone treatment (30 mg/kg twice a day for seven days).18 The recurrent parasites showed less sensitivity to atovaquone than those obtained before treatment.18
Babesia gibsoni DNA was isolated from blood samples using a genomic DNA extraction kit (GFX Genomic Blood Purification Kit; Amersham, Buckinghamshire, United Kingdom). Total RNA from B. gibsoni was isolated using ISOGEN (Nippon Gene, Toyama, Japan) according to the manufacturer’s instructions. Reverse transcription to obtain cDNA was performed as follows: 10 µg of total RNA denatured at 65°C was reverse transcribed in a total volume of 40 µL using 2 µg of oligo-dT primer and 200 units of Superscript II reverse transcriptase (TaKaRa, Shiga, Japan) in a solution containing 50 mM Tris-HCl, pH 8.3, 3 mM MgCl2, and 75 mM KCl (RT buffer) with 100 µM of each dNTP at 42°C for 1 hr.
The primer pair was designed according to the sequence of nucleotides from B. bovis (GenBank accession no. AF053002), B. bigemina (accession no. F109354),21 and Theileria annulata (accession no. M63015)22 as follows: JD279, 5'-TGG AA(C/T) TT(A/T) GGG TTT-3' and ROR8BG, 5'-A(A/T)G G(A/T)A TTA CTC CAT AAG TTA-3' . A polymerase chain reaction (PCR) was performed on 20 µL of a mixture containing 1 µg of template genomic DNA, 10 pmol of each primer, 200 µM deoxynucleoside triphosphate (dNTP), and 1.25 units Taq Gold DNA polymerase (Invitrogen, Carlsbad, CA). The PCR was repeated for 40 cycles with denaturation for 30 seconds at 94°C, annealing for 1 minute at 40°C, and extension for 1 minute at 72°C to obtain a 533-basepair fragment. The PCR product was ligated into the pCR2.1-TOPO vector using the TOPO TA Cloning kit (Invitrogen). The entire ligation reaction was used to transform Escherichia coli DH5
competent cells. Plasmid DNA from two positive transformants was used to sequence the DNA of the insert. Both strands of the plasmid insert DNA were sequenced using the Dye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA). The sequencing analysis was performed using GENETYX-MAC version 10 (Software Development, Co. Ltd., Tokyo, Japan). The similarities of this nucleotide sequence to those of B. bovis, B. bigemina, and T. annulata were 82.2%, 80.3%, and 64.5%, respectively.
Subsequently, 3' rapid amplification of cDNA ends was performed to determine the sequence of the gene at the 3' end of cytb. A primer was designed as follows: Ky1, 5'-GAG TAT TAA CAG AAG TTA ATA TGG-3'. We performed a PCR on 1 µL cDNA using 50 pmol of primer and 2.5 units of Taq polymerase (TaKaRa) in a solution containing 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl2, 50 mM KCl, 0.01% gelatin, and 200 mM of each dNTP. The thermal cycling conditions were one cycle for 5 min at 94°C; five cycles for 1 minute at 94 °C, 48°C, and 72°C; and a final incubation for 10 minutes at 72°C. The resulting 670-basepair fragment was cloned using the method described earlier into the pCR2.1-TOPO vector and the sequence was then determined.
A commercial primer kit (DNA Walking Speed up kit; See-gene, Del Mar, CA) was used to sequence the 5' end of cytb, and three primers were designed corresponding to the sequences of the cytb fragments obtained from JD279 and ROR8BG: Ky4, 5'-CAG GTT TGT TAT AAC TGT TGC TCC-3' , Ky6, 5'-ACG AAC TGC CCA ACC CAT AT-3' , and Ky2, 5'-CCC ATA TTA ACT TCT GTT AAT ACT C-3' . A primer set for CYTb 1, 5'-TGT ATT ACT ATA CTG TGA GT-3' and CYTb 2, 5'-AAC TCC CCT CTG TTT TA- 3' , was designed corresponding to the nucleotide sequences obtained for the 3' and 5' ends (Figure 1
). The PCR was conducted using primers CYTb 1 and CYTb 2 with B. gibsoni genomic DNA and the resulting fragment of 1,228 basepairs was cloned and sequenced as described earlier in this report.
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). The ORF shows a highly conserved protein with strong identity throughout to that of other apicomplexan parasite cytb amino acids. Comparison of the deduced amino acid sequence against those of B. bovis, B. bigemina, and T. annulata, which are partially defined,21,22 showed 79.7%, 82.5%, and 52.1% identity, respectively, within their defined partial sequences. Comparison of the deduced amino acid sequence against those of Theileria parva (accession no. Z23263), P. falciparum (accession no. AU086218), and T. gondii (accession no. AF023246) showed 53.8%, 41.4%, and 41.5% identity, respectively (Figure 2). The nucleotide sequence of the cytb gene amplified from blood samples taken from two dogs in Okinawa Prefecture19 showed > 99% identity to that obtained from dogs in Aomori Prefecture. At amino acid 309, tyrosine was replaced by cysteine in B. gibsoni isolated in Okinawa Prefecture.
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).23 Moreover, the same substitution has been reported in atovaquone-resistant Plasmodium spp.13,23 Since the recurrent parasites in this study displayed less atovaquone sensitivity after atovaquone treatment than in our previous study,18 the M121I mutation identified in this study may be associated with the loss of sensitivity.
Two other single polymorphisms, a G-to-A substitution at nucleotide 658, which resulted in valine being replaced by isoleucine (V220I mutation), and an A-to-G substitution at nucleotide 907, which resulted in isoleucine being replaced by valine (I303V), were observed in the cytb gene of B. gibsoni from two of three dogs after atovaquone treatment. The V220I mutation was a mixed population with wild-type nucleotide in B. gibsoni from dog B and dog C, but was not detected in B. gibsoni from dog D. The I303V mutation was a complete substitution in B. gibsoni from dog B, a mixed population with wild-type nucleotide in B. gibsoni from dog C, and was not detected in dog D (Figure 3). Whether the polymorphisms induce less sensitivity against atovaquone and how sensitivity is reduced in B. gibsoni are not known. Further study is needed to establish a direct correlation between the three single polymorphisms and atovaquone resistance.
Direct sequencing in B. gibsoni DNA extracted from pre-treatment dogs and of parasite DNA extracted from the Okinawa Prefecture isolate, which had not been exposed to atovaquone, failed to detect these three nucleotide polymorphisms. Additional screening of B. gibsoni isolated from two naturally infected Tosa dogs in Aomori Prefecture that had not been treated with atovaquone also showed that these polymorphisms were absent. A single seven-day course of atovaquone treatment might induce changes in the population of B. gibsoni with variant polymorphisms.
Atovaquone is a major component of a new antibabesial treatment in which azithromycin is used as a combination drug for canine babesiosis.17 This information on polymorphisms of the atovaquone-binding site in B. gibsoni may be useful not only for treatment of canine babesiosis, but also for treatment of human babesiosis. These single polymorphisms in the cytb gene may be useful as molecular markers for monitoring the development and spread of drug-resistant parasites in the field. We are presently conducting an in vitro study to establish whether atovaquone resistance is associated with the three polymorphisms in the cytb gene.
Received June 27, 2005. Accepted for publication September 13, 2005.
Financial support: This study was supported by Grants-in-Aid for Scientific Research and Young Scientists from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Japan Society for the Promotion of Science, and the Kitasato University Research Grant for Young Researchers.
* Address correspondence to Aya Matsuu, Department of Small Animal Internal Medicine 1, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034-8628, Japan. E-mail: matsuu{at}umas.kitasato-u.ac.jp 
Authors’ addresses: Aya Matsuu, Kayoko Miyamoto, and Seiichi Higuchi, Department of Small Animal Internal Medicine 1, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034-8628, Japan. Hiromi Ikadai, Department of Veterinary Parasitology, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034-8628, Japan. Shozo Okano, Department of Small Animal Surgery 3, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034-8628, Japan. Telephone: 81-176-23-4371, E-mail: matsuu{at}umas.kitasato-u.ac.jp
Reprint requests: Aya Matsuu, Department of Small Animal Internal Medicine 1, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034-8628, Japan, Telephone: 81-176-23-4371 ext 321, E-mail: matsuu{at}vmas.kitasato-u.ac.jp.
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(V220I), and 
(I303V).