Veterinary Parasitology 106 (2002) 265–271

Short communication Analysis of the 18S rRNA gene sequence of a detected in two Japanese dogs Hisashi Inokuma a,∗, Masaru Okuda a, Koichi Ohno b, Kazunobu Shimoda c, Takafumi Onishi a a Laboratory of Veterinary Internal Medicine, Faculty of Agriculture, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan b Graduate School of Agriculture and Life Science, The University of Tokyo, Tokyo 113-8657, Japan c Shimoda Animal Hospital, Nohgata, Fukuoka 822-0007, Japan Received 25 September 2001; received in revised form 20 March 2002; accepted 20 March 2002

Abstract Partial sequences of the 18S rRNA gene (625 bp) from a Hepatozoon detected in two canine hep- atozoonosis cases, one clinical and one subclinical, in Japan were analyzed. Both sequences were identical to each other and they were closely related to the Hepatozoon canis strain found in Israel with 99% (617/625) nucleotide identity. Both Hepatozoon americanum and Hepatozoon catas- bianae were distantly related to the Japanese Hepatozoon with 94% (586/625) and 91% (566/625) identities, respectively. In a phylogenetic tree, the Japanese Hepatozoon was most closely related to H. canis from Israel but was significantly different than H. americanum and H. catasbianae. These results suggest that the Hepatozoon detected in the Japanese dogs might be a strain variant of H. canis, but is apparently a different species than H. americanum. © 2002 Elsevier Science B.V. All rights reserved.

Keywords: Dogs; Hepatozoon; Japan

1. Introduction

Canine hepatozoonosis is a tick-borne protozoan disease caused by two known pathogens: Hepatozoon americanum and Hepatozoon canis. The geographic distribution, vectors and pathogenesis of these two pathogens are reported to be different. H. americanum is a causative agent of canine hepatozoonosis in the US, which is transmitted by Amblyomma

∗ Corresponding author. Tel.: +81-83-933-5895; fax: +81-83-933-5895. E-mail address: [email protected] (H. Inokuma).

0304-4017/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S0304-4017(02)00065-1 266 H. Inokuma et al. / Veterinary Parasitology 106 (2002) 265–271 maculatum ticks (Vincent-Johnson et al., 1997b). The agent causes a distinct clinical syn- drome in dogs characterized by fever, lethargy, weight loss, stiffness, signs of pain, paralysis, and ocular discharge (Vincent-Johnson et al., 1997a,b; Macintire et al., 2001). Diagnosis of American hepatozoonosis is made based on a muscle biopsy to detect the pathogens (Craig et al., 1984). H. canis is the cause of Old World canine hepatozoonosis reported from southern Europe, the Middle East, Africa and the Far East. The main vector of the disease is the brown dog tick, Rhipicephalus sanguineus (Baneth et al., 2001). Pathogene- sis of H. canis is thought to be weak, because subclinical infections are common, usually causing a milder disease that affects the spleen, lymph nodes, and bone marrow, resulting in anemia and lethargy (Baneth and Weigler, 1997). Gametocytes in circulating white blood cells are often an incidental finding in dogs without clinical signs and the disease is easily diagnosed by observation of the blood film (Baneth et al., 1996). Both clinical and subclinical cases of canine hepatozoonosis have been reported in Japan since 1991. Most cases in Japan are subclinical with gametocytes appearing in the periph- eral blood (Murata et al., 1993a,b,c). These findings are similar to those of cases reported from Israel (Baneth et al., 1996, Baneth and Weigler, 1997). On the other hand, clinical cases with lethargy, weight loss, obvious signs of pain, leukocytosis or periosteal new-born proliferation were also reported similar to the American type of hepatozoonosis (Makimura et al., 1991, Murata et al., 1991, Nakayama et al., unpublished data). Furthermore, Haema- physalis spp. is suspected to be the vector tick of canine hepatozoonosis in Japan (Murata et al., 1995). Given this evidence, one may suspect that the pathogen of the Japanese hep- atozoonosis is different from that of USA and Israel. Although genetic analysis revealed that Hepatozoon strains found in the US and Israel are quite different (Baneth et al., 2000), no genetic analysis has been done for the pathogen of canine hepatozoonosis in Japan. Thus, we analyzed the partial 18S rRNA gene sequences of Hepatozoon detected in two canine cases in Japan, one clinical and the other subclinical, to clarify the phylogeny of the Japanese Hepatozoon in dogs.

2. Materials and methods

Two canine cases of Hepatozoon infection were used in this study. The first case was a clinical hepatozoonosis. A male Beagle, 1 year and 9 months old, born in Fukuoka Prefecture was admitted to animal hospital in Yamaguchi University for stiffness, paralysis, depression and anorexia on 21 March 1997 (day 1). The mother was also infected with Hepatozoon without clinical signs. The second case was a mongrel dog, 1 year and 6 months old, in Yamaguchi Prefecture. The dog was presented at the animal hospital in Yamaguchi University for firalia control in May 1998. No clinical signs were found in the second case. PCR and sequencing were performed with EDTA-anticoagulated peripheral blood taken on the first day for both cases. The buffy coat of each blood sample was separated and kept frozen at −80 ◦C until used. DNA from each sample was extracted following the QIAamp Tissue Kit procedure (QIAGEN GmbH, Hilden, Germany). Finally each DNA sample was eluted in 200 ␮l of TE buffer. A primer set, HepF (5 ATA-CAT-GAG-CAA-AAT-CTC-AAC 3) and HepR (5 CTT-ATT-ATT-CCA-TGC-TGC-AG 3), was designed to amplify a partial 18S rRNA gene sequence of Hepatozoon spp. based upon alignment data from the H. canis H. Inokuma et al. / Veterinary Parasitology 106 (2002) 265–271 267 from a dog in Israel (GenBank accession number AF176835), H. americanum from a dog in the US (AF176836) and H. catesbianae from a frog (AF176837). PCR conditions were as previously described (Inokuma et al., 2001) with an annealing temperature of 57 ◦C. Five millilitre of each DNA was used as the template. The positive amplification product with 666 base pairs was then extracted using the QIA PCR purification kit (QIAGEN GmbH) for sequence analysis. The fluorescence-labeled dideoxynucleotide method was used for DNA sequencing reactions (Perkin-Elmer, Applied Biosystems Division, Foster City, CA). Sam- ples were then sequenced using a Perkin-Elmer ABI Prism 377 automated DNA sequencer at The DNA Core Facility of the Center for Gene Research, Yamaguchi University. The nucleotide sequence was confirmed by two independent PCR amplification and sequenc- ing experiments. The sequences of Hepatozoon Japan and the registered sequences of other related protozoal species deposited in GenBank were analyzed for phylogenetic re- lationships. The GenBank accession numbers of the 18S rRNA gene sequences used to construct phylogenetic trees are as follows: peromysci, AF339492; vivax, U93233; cruzi, AF017120; caninum, U16159; , L37415; parvum, AF115377; parva, L02366; gibsoni, AF158702 and Trypanosoma cruzi, AF359496. Multiple alignment analysis, dis- tance matrices calculation, and construction of a phylogenetic tree were performed with the ClustalW program (Thompson et al., 1994) version 1.8 in the DNA Data Bank of Japan (DDBJ; Mishima, Japan). The distance matrices for the aligned sequences with all gaps ignored were calculated using the Kimura two-parameter method (Kimura, 1980), and the neighbor-joining method was used for constructing a phylogenetic tree (Saitou and Nei, 1987). The stability of the tree obtained was estimated by bootstrap analysis for 100 replications using the same program. Tree figures were generated using the Tree View pro- gram version 1.61 (Page, 1996). The sequence of the partial 18S rRNA gene of Japanese Hepatozoon determined in this study has been deposited in GenBank data library under accession number AF418558.

3. Results and discussion

Physical examination of the first case revealed a swelling of the hind legs, signs of pain and mild dehydration, but no fever was observed (38.7 ◦C). Results of hematological and biochemical analysis on the same day indicated nonregenerative anemia (red blood cell counts 312 × 104/ml, packed cell volume 19%, hemoglobin concentration 6.9 g/dl, reticu- locytes 0.7%), mild leukocytosis (21,500/␮l), thrombocytopenia (21,000/␮l), high activities of serum alkaline phosphatase (1083 IU/l) and creatine kinase (834 IU/l), hypoalbuminemia (2.1 g/dl) and a high concentration of serum C-reactive protein (CRP, 8.4 mg/dl). Gameto- cytes were found in 11% of neutrophil-like cells in peripheral blood stained with Giemsa (Fig. 1). A muscle biopsy was performed, but nothing significant was found in the hind legs or hip. Radiographic findings showed periosteal new-borne proliferation at the bilat- eral humerus and femur. Treatment with toltrazuril, imidocarb, diminazene aceturate and clindamycin did not improve the clinical signs. Prednisolone was also used occasionally to control the pain and inflammation suspected to be due to the high CRP levels. All treatment had been stopped at the end of October 1997, when the gametocytes in peripheral blood 268 H. Inokuma et al. / Veterinary Parasitology 106 (2002) 265–271

Fig. 1. Gametocytes found in neutrophil-like cells of the buffy coat of peripheral blood taken from case no. 1. Giemsa’s stain (×1000). disappeared, but the stiffness, signs of pain, and lethargy continued. The patient died of unknown causes in the summer of 1999, and post-mortem examination was not performed. In the second case, 3% of the white blood cells were infected with Hepatozoon gametocytes, results of blood examination were within the normal ranges, and radiographic findings in all the legs were also normal. Canine hepatozoonosis is an endemic disease in the western part of Japan (Murata et al., 1993a, Inokuma et al., 1999). There have been other clinical cases like case no. 1 in the present study, which showed lethargy, stiffness, signs of pain and paralysis (Makimura et al., 1991, Murata et al., 1991). These findings were similar to those of H. americanum infection (Vincent-Johnson et al., 1997a, b, Macintire et al., 1997, 2001). Nonregenerative anemia, substantial leukocytosis, hypoalbuminemia and increased serum activity of alkaline phos- phatase were also recorded as hematologic abnormalities in the American hepatozoonosis (Macintire et al., 1997, 2001). New-borne proliferation was also found in the radiographic findings (Macintire et al., 1997). On the other hand, most of the cases in Japan are subclinical with numbers of gametocytes appearing in peripheral blood as in case no. 2. These findings are similar to findings in H. canis infection reported as Old World canine hepatozoonosis, which usually causes a milder disease (Baneth et al., 1996, Baneth and Weigler, 1997). The Hepatozoon detected in Japanese dogs cannot be identified by its pathogenesis, vectors or morphological studies. Thus molecular analysis was performed to clarify the phylogeny of the Japanese Hepatozoon. A positive PCR signal was detected with the DNA extracted from each dog. Analyzing the nucleotide sequences of the 625 bp PCR product excluding the primer regions revealed that both sequences were 100% identical to each other. Pairwise percent identities of the H. Inokuma et al. / Veterinary Parasitology 106 (2002) 265–271 269 270 H. Inokuma et al. / Veterinary Parasitology 106 (2002) 265–271 sequences with all gaps omitted were calculated. The sequence of the Japanese Hepatozoon was similar to that of H. canis from Israel with 99% (617/625) nucleotide identity. Both H. americanum and H. catasbianae were distantly related to Japanese Hepatozoon with 94% (586/625) and 91% (566/625) identities, respectively. The phylogenetic tree is shown in Fig. 2. All the Hepatozoon species analyzed in the present study were in the same group. Hepatozoon detected from dogs in Japan was the most closely related to H. canis in Israel, but showed significant distance from both H. americanum and H. catasbianae. These results suggest that Hepatozoon detected in Japanese dogs might be a strain variant of H. canis, but is apparently a different species from H. americanum. The suspected vectors of Japanese Hepatozoon are Haemaphysalis ticks (Murata et al., 1995), which are different from the vector ticks of H. americanum and H. canis. Analyses of other gene sequences and antigens were needed to explain the differences in the pathogenesis and vectors of Hepatozoon in other countries. As partial sequences (625 bp) of the 18S rRNA gene of the agent from only two dogs were analyzed in the present study, pathogens from more dogs should be analyzed over longer sequences of the gene to confirm the results in this study. Furthermore, many western breeds of dogs are imported to Japan from other countries, including the USA where H. americanum infection is dominant. It is suspected that H. americanum also exists in Japan, though the vector tick A. maculatum has never been found there. More epidemiological studies using molecular tools are also required to clarify the canine hepatozoonosis in Japan.

Acknowledgements

The authors would like to acknowledge the technical expertise of the DNA Core facility of the Center for Gene Research, Yamaguchi University, supported by a grant-in-aid from the Ministry of Education, Science, Sports and Culture of Japan. We also acknowledge Dr. M. Terado for her technical assistance in the treatment and care of the patients, and thank Dr. M. L. B. Montemayor for English correction.

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