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Detection and analysis of Babesia species from ticks recovered from dogs in Japan were attempted by PCR and nucleotide sequence analysis based on the 18S rRNA gene, respectively. A total of 1,136 ticks were examined for Babesia DNA by 18S rRNA-based PCR and nucleotide sequencing. Partial sequences of Babesia canis vogeli DNA were detected from six ticks in Aomori, Nara, Hiroshima, Oita, and Okinawa Prefectures; and Babesia gibsoni Asia-1 DNA was also detected in four ticks in Osaka, Hiroshima, Miyazaki, and Okinawa Prefectures. Unique sequences of 1,678 bp were also obtained from Ixodes ovatus ticks in Akita and Fukui Prefectures. The sequences were similar to those of Babesia odocoilei (97.7%) and Babesia divergens (97.6%). This is the first report of the detection of DNA belonging to this group in Japan.
Animals are often exposed to a large number of tick species, depending upon the distribution of these arthropod vectors in the environment. Recently, interest in ticks of domestic animals has been increasing because of emerging and reemerging tick-borne diseases, including those caused by rickettsial, bacterial, and protozoal pathogens, and their zoonotic nature. Ixodid tick species from canine hosts in Japan were documented in a recent report (19). The tick species of canine hosts in Japan showed more variation than the tick species of canine hosts in European countries. Haemaphysalis longicornis was the species most frequently found. This was followed by Hamaphysalis flava, Rhipicephalus sanguineus, and Ixodes ovatus as the next most dominant tick species. As dogs are in close contact with humans, they are possible carriers of tick vectors to the human environment. Babesia species are among the major tick-borne pathogens that infect the red blood cells of humans and animals worldwide. Three Babesia species affecting humans and dogs have been reported in Japan. Babesia microti-like parasites were first documented in Japanese field mice in 1984 (20), and recently, a human case of B. microti-like infection has been reported (15). This B. microti-like protozoon has a wide geographical distribution, ranging from northern to western Japan; however, neither the reservoir nor the vector has been thoroughly investigated (23). There are no reports on the relationship between dogs and B. microti-like protozoa. Both Babesia canis and Babesia gibsoni are well-known Babesia species in canine hosts (27). However, there have been few epidemiological studies on the distribution of the Babesia species of canine hosts in Japan.
Recently, molecular techniques including PCR and sequence analysis have been used for the epidemiological study and phylogenetic analysis of piroplasmas (2, 4, 8, 13). The advantage of molecular methods over other techniques are their sensitivities for the detection of Babesia protozoa in peripheral blood and arthropod vectors. Furthermore, subsequent sequence analysis provides phylogenetic information on the pathogen. Because blood-sucking vectors contain infected host blood and the pathogen itself, analysis of these vectors is a reliable tool with which to demonstrate the existence of pathogens in a specific area (17). Ticks have also been used for the epidemiological study of tick-borne pathogens (21). Thus, in the present study, the detection and analysis of Babesia species from ticks recovered from dogs in Japan were attempted by using molecular methods, including PCR and sequence analysis of the 18S rRNA gene.
A total of 4,122 ticks were recovered from 1,221 domestic dogs from 47 prefectures in Japan and were stored in 70% ethanol for morphological identification (19). After identification, one tick per dog was selected for screening analysis. All the ticks selected were semiengorged or fully engorged adult females or nymphs. Total DNA was extracted from each tick by using the QIAamp DNA Mini kit (QIAGEN GmbH, Hilden, Germany), placed in 200 μl of TE (Tris-EDTA) buffer, and stored at −20°C until further use. The success of DNA extraction was confirmed by PCR with a primer set consisting of primer 28SF (GAC-TCT-AGT-CTG-ACT-CTG-TG) and primer 28SR (GCC-ACA-AGC-CAG-TTA-TCC-C) for detection of the 28S rRNA genes of the ticks. These primers were designed on the basis of the alignment data for the 28S rRNA gene sequences of Hamaphysalis, Rhipicephalus, and Ixodes.
For screening purposes, PCR amplification was performed with a 25-μl reaction mixture containing 5 μl of each DNA template with a set of primers, primer Babesia-F (GTG-AAA-CTG-CGA-ATG-GCT-CA) and primer Babesia-R (CCA-TGC-TGA-AGT-ATT-CAA-GAC), which were designed on the basis of the common sequence of the 18S rRNA gene of the genus Babesia. The amplification procedure was performed as reported previously (10). To analyze the infecting species, an approximately 650-bp PCR product from a positive reaction was purified by using the QIAPCR purification kit (QIAGEN) for direct sequence analysis with a Perkin-Elmer ABI Prism 377 automated DNA sequencer at the DNA Core Facility of the Center for Gene Research, Yamaguchi University. The sequence data for the PCR products were analyzed by using the BLAST program (version 2.0; National Center for Biotechnology Information [http://www.ncbi.nlm.gov/BLAST/]) for the detection of homologous sequences. If the sequence did not show 100% homology with sequences registered in GenBank, an analysis with a longer sequence of the 18S rRNA gene was attempted with amplification primers RIB19 and RIB20 (28) and the following sequencing primers: Seq F1 (AGT-AGT-CAT-ATG-CTT-GTC), Seq F2 (CCG-TGC-TAA-TTG-TAG-GGC-TA), Seq F3 (CAG-AGT-ATC-AAT-TGG-AGG-GC), Seq F4 (CGA-TCA-GAT-ACC-GTC-GTA-G), Seq F5 (CTT-AGA-GGG-ACT-TTG-CGG), SeqR1 (CTT-CCT-TTA-AGT-GAT-AAG-GTT-CAC), SeqR2 (TCG-ATG-GAC-GCA-TCA-GTG), and SeqR3 (GTC-AGG-ATT-GGG-TAA-TTT-GC).
The sequences of the 18S rRNA genes determined were analyzed for their phylogenetic relationships with other sequences registered in GenBank. Multiple-sequence-alignment analysis, determination of the pairwise percent identities of the sequences, distance matrix calculations, and the construction of phylogenetic trees were all performed with the ClustalW program (version 1.8) (22) in the DNA data bank of Japan (DDBJ; Mishima, Japan [http://www.ddbj.nig.ac.jp/htmls/E-mail/clustalw-e.html]), as described in a previous report (10). The distance matrices for the aligned sequences, with all gaps ignored, were calculated by the Kimura two-parameter method (12), and the neighbor-joining method was used to construct a phylogenetic tree (18). The stability of the tree obtained was estimated by bootstrap analysis with 100 replications by using the same program. The phylogenetic trees were generated by using the Tree View program (version 1.61) (16).
Nucleotide sequence accession numbers. The GenBank accession numbers of the 18S rRNA gene sequences of other species used to analyze the data are as follows: Babesia divergens, U16370; Babesia odocoilei, U16369; B. gibsoni Asia-1, AF175300; B. gibsoni Asia-2, AF175301; B. canis vogeli, AY072925; B. canis canis, AY072926; Babesia caballi, Z15104; Babesia bigemina, X59607; Babesia ovata, AY081192; Babesia microti Kobe, AB050732; and Theileria sergenti Ikeda, AB000271. The nucleotide sequences of the 18S rRNA genes of the Babesia species detected from ticks 610 and 615 in Akita Prefecture and tick 766 in Fukui Prefecture have been deposited in the GenBank database under accession numbers AY190123, AY190125, and AY190124, respectively.
DNA was successfully extracted from 1,136 of 1,221 ticks examined. A total of 13 tick samples showed a single band of the appropriate size by the screening PCR. The dogs infected with these particular ticks did not show any clinical signs of Babesia infection, such as fever, anorexia, anemia, or jaundice. The peripheral blood of these dogs could not be analyzed in this study. By analyzing the sequences of the 608-bp PCR products, excluding the primer region, six ticks in Aomori, Nara, Hiroshima, Oita, and Okinawa Prefectures were identified as B. canis vogeli (GenBank accession no. AY072925) (Fig. (Fig.11 and Table Table1).1). The other four ticks, in Osaka, Hiroshima, Miyazaki, and Okinawa Prefectures, were found to be identical to B. gibsoni Asia-1 (GenBank accession no. AF175300) (Fig. (Fig.11 and Table Table1).1). The other three I. ovatus females (ticks 610 and 615 in Akita Prefecture and tick 766 in Fukui Prefecture) were found to be closely related to B. odocoilei (GenBank accession no. U16369) or B. divergens (GenBank accession no. U16370), with identities of 96.4 and 95.4%, respectively (Fig. (Fig.11 and Table Table1).1). Female I. ovatus tick 611, recovered from the same dog as I. ovatus tick 610, was also analyzed for Babesia infection by PCR, but a negative result was obtained.
To confirm the results of the screening PCR, determination of a longer 18S rRNA gene sequence of three ticks (ticks 610, 615, and 766) was attempted with amplification primers RIB19 and RIB20, which produce products of approximately 1,700 bp. The 1,678-bp nucleotide sequence, excluding the primer region, successfully obtained from ticks 610 and 766 was similar to the sequences of B. odocoilei and B. divergens, with identities of 97.7 and 97.6%, respectively. The percent identities of this Babesia sp. detected from I. ovatus ticks are summarized in Table Table2.2. The phylogenetic analysis showed that the Babesia sp. detected from I. ovatus belongs to the same group as B. odocoilei and B. divergens (Fig. (Fig.22).
In the present study, ticks recovered from canine hosts were used for the epidemiological study of dog-related Babesia species in Japan. One tick was selected from each dog for screening purposes. All the ticks selected were fully engorged or semiengorged adults or nymphs, meaning that they contained peripheral blood of the canine host. Thus, a positive PCR result would lead to two possible sources of Babesia infection, either the tick or the dog.
Both B. canis and B. gibsoni are well-known Babesia species with canine hosts in Japan. B. canis is believed to be distributed only in subtropical Okinawa, in accordance with the distribution of its tick vector, R. sanguineus (27). Indeed, one R. sanguineus tick in Okinawa showed B. canis vogeli infection. However, recent studies revealed that this species has established itself in mainland Japan (11, 19). In the present study, the DNA of B. canis vogeli was also detected from Haemaphysalis ticks recovered from dogs in Aomori, Nara, Hiroshima, and Oita Prefectures. However, R. sanguineus was not detected on these dogs. These dogs might have previously been infected with B. canis. R. sanguineus was thought to have been introduced into Japan by U.S. army personnel (11). There are several U.S. military bases in Japan, including Aomori, Tokyo, Kanagawa, Yamaguchi, and Okinawa Prefectures. The fact that the DNA of B. canis, a tropical type of Babesia species, has been detected in Aomori Prefecture, in northern Japan, might be related to the U.S. military base in that Prefecture. Because of the movement of dogs, humans, and materials both nationally and internationally, the distribution of the tick vectors of Babesia parasites is variable. B. gibsoni is believed to be distributed mainly in western Japan. H. longicornis, the main vector tick of B. gibsoni, has a wide geographical distribution and is the most dominant tick species on canine hosts (19). However, in the present study, positive results were obtained for only four ticks, which were found in Osaka, Hiroshima, Miyazaki, and Okinawa Prefectures, all of which are in western Japan. No ticks in eastern Japan were positive for B. gibsoni.
I. ovatus ticks recovered from three dogs in Akita and Fukui Prefectures had 18S rRNA gene nucleotide sequences closely related to the 18S rRNA gene nucleotide sequence of B. odocoilei or B. divergens. Two I. ovatus females were recovered from one of these dogs in Akita Prefecture, but only one tick was positive for Babesia DNA. The result suggests that the tick was infected with the Babesia sp., while the dog was not. This newly found Babesia sp. may be associated with I. ovatus ticks. Sequence analysis of the 18S rRNA gene suggested that this Babesia sp. detected in I. ovatus might be new. It marks the first time that this group of Babesia has been detected in Japan. Both B. odocoilei and B. divergens infect ruminants and are transmitted by Ixodes spp. B. odocoilei infects white-tailed deer in North America, and its pathogenic potential is believed to be moderate to weak (7, 24, 25). The tick vector of B. odocoilei is Ixodes scapularis (24). B. divergens is found in European countries and shows a mild to moderate pathogenic potential in bovine and ovine hosts (3, 14). The tick vector of B. divergens is Ixodes ricinus (6). Thus, one may suspect that any Babesia sp. closely related to B. divergens and B. odocoilei would be pathogenic for ruminants. More epidemiological studies are needed to clarify the vectors, host animals, reservoirs, and distribution of this Babesia species in Japan. Furthermore, clinical studies of humans infected with this Babesia species are required, because B. divergens is known to be a zoonotic pathogen and several cases of B. divergens infection in humans have been reported, mainly in European countries (1, 5, 9).
Recent research revealed that B. microti-like protozoa associated with rodents showed a wide distribution, ranging from northern to western Japan (23, 26); however, this Babesia species was not detected in the present study. The distributions of B. microti-like protozoa and Babesia spp. associated with canine ticks might be different.
In addition, this study demonstrated that the combination of PCR and sequence analysis is useful for the epidemiological study of ticks recovered from animals.
We acknowledge the technical expertise of the DNA Core Facility of the Center for Gene Research, Yamaguchi University. We also thank Nippon Zenyaku Kogyo Co., Ltd., for collecting and sending the tick samples.
The DNA Core Facility of the Center for Gene Research, Yamaguchi University, is supported by a grant-in-aid from the Ministry of Education, Science, Sports and Culture of Japan. This work was supported in part by Merial Japan Ltd. and a grant-in-aid (grant 14360190) for Scientific Research from the Japan Society for the Promotion of Science.