Journal of Wildlife Diseases, 40(4), 2004, pp. 804–807 ᭧ Wildlife Disease Association 2004

Leucocytozoon lovati Infections in Wild Rock Ptarmigan (Lagopus mutus) in Japan

Mio Hagihara,1 Tsuyoshi Yamaguchi,2 Masanobu Kitahara,3 Katsuya Hirai,2 and Koichi Murata1,4 1 Depart- ment of Wildlife Science, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kana- gawa 252-8510, Japan; 2 Department of Veterinary Microbiology, Gifu University, 1-1 Yanagito, Gifu 501-1112, Japan; 3 Institute of Alpine Environmental Research, 12-388 Inao, Taira, Ohmachi, Nagano 398-0001, Japan; 4 Corresponding author (email: [email protected])

ABSTRACT: Leucocytozoon lovati infections meadows, and the tundra of the Northern were detected in free-flying rock ptarmigan Hemisphere (Haneda et al., 1985). The (Lagopus mutus), an endangered species that inhabits alpine areas in Japan. Eight of nine habitat in Japan is the alpine zone in the adult birds tested positive for L. lovati infec- central part of the main island, where the tion. For comparison, two captive rock ptar- birds inhabit regions at 1,800–3,000 m el- migans hatched in a breeding facility at the foot evation in the summer (May–August) and of the mountains were examined. Both were fly to forest areas at lower elevations from negative for L. lovati infection. This is the first report of L. lovati infection in the rock ptar- winter to early spring (September–April). migan in Japan. The present population is estimated at Key words: Hematozoa, Japan, Lagopus about 3,000 birds and the species is listed mutus, Leucocytozoon lovati, rock ptarmigan. as vulnerable in the Appendix of the Law for Conservation of Endangered Species Leucocytozoon spp. are hematozoan of Wild Fauna and Flora and also desig- parasites that are transmitted by blood- nated as a protected species of one of the sucking arthropods (Jordan, 1990). Cur- Special Natural Monuments under the rently, more than 60 species of Leucocy- Law for the Protection of Cultural Prop- tozoon have been detected from birds erties in Japan. throughout the world (Bennett et al., Currently nothing is known about the 1994). Although it is rare that leucocyto- blood parasites of rock ptarmigan in Japan. zoonosis is incriminated as a cause of The present study was undertaken to de- death, it has been observed in some avian termine the presence and prevalence of L. species, including waterfowl (Greiner, lovati in populations of rock ptarmigan in 1991), eagles (Stuht et al., 1999), and two areas on the main island in Japan. grouse (Clarke, 1935). Leucocytozoon lov- Rock ptarmigan were captured with the ati is a common parasite of birds belonging permission of the Ministry of the Environ- to the Tetraoninae and has a Holarctic dis- ment of the Japanese Government on Mt. tribution (Bennett et al., 1991). Infections Tateyama (36Њ35ЈN, 137Њ36ЈW) at about have been reported in red grouse (Lago- 2,400 m elevation in April and on Mt. Jii- pus lagopus scoticus), sage grouse (Centro- gatake (36Њ35ЈN, 137Њ45ЈW) at about 2,650 cercus urophasianus), ruffed grouse (Bon- m elevation in June 2002. The birds were asa umbellus), black grouse (Tetrao tetrix), captured in the daytime by using a long blue grouse (Dendragapus obscurus), fishing rod with a looped soft wire fixed at western capercaillie (Tetrao urogallus), the tip. The birds were humanely re- willow ptarmigan (Lagopus lagopus), as strained by hand to check the band num- well as rock ptarmigan (Lagopus mutus) ber and confirm body condition before (Sambon, 1908; Fantham, 1910; Clarke, withdrawing a blood sample. Two adult fe- 1935; Stabler et al., 1967; Bennett et al., males and three adult males were sampled 1991; Forbes et al., 1994; Dunbar et al., in April and four adult males were sam- 2003; Holmstad et al., 2003). pled in June. All individuals were deter- The rock ptarmigan is adapted to a wide mined to be in good health by visual ex- range of natural habitats such as forests, amination. For comparison, two captive

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FIGURE 1. Photomicrographs of gametocytes and the host-cell complex of Leucocytozoon lovati from the rock ptarmigan (Lagopus mutus) in Japan. Macrogametocytes are shown in A–D and microgametocytes are shown in E–H. Barϭ10 ␮m. adult male rock ptarmigans (3 and 8 yr The stained blood films were observed old) kept in a captive-breeding facility at under a light microscope and about 400 the Ohmachi Alpine Museum (36Њ30ЈN, leucocytes were observed on each slide. 137Њ52ЈW), City of Shinano Ohmachi, Na- The ratio of infected cells to noninfected gano Prefecture, at 880 m elevation were cells was calculated, and the intensity of examined in October 2001 and 2002. parasitism was classified by the Ashford Peripheral blood was collected from the scale (Ashford et al., 1990). Parasitism was wing vein by using sodium heparin as an scored on a scale of 0–4 as follows: 0, no anticoagulant. The heparinized blood sam- parasites in 100 fields under low magnifi- ples were transported in a cooler box (4 cation (100ϫ); 1, one parasite; 2, 2–10 par- C) to our laboratory at Nihon University, asites; 3, 11–100 parasites; and 4, 100 or Kanagawa Prefecture, where hematologic more parasites in 100 fields under high examinations were performed. Thin blood magnification (400ϫ). films were made, fixed with 99.5% ethanol, The morphology of micro- and macro- and stained with HEMACOLOR௡ (Merck gametocytes was studied under oil immer- Co., Darmstadt, Germany) in the labora- sion (1,000ϫ), and measurements were tory. made in micrometers. The width and 806 JOURNAL OF WILDLIFE DISEASES, VOL. 40, NO. 4, OCTOBER 2004

TABLE 1. Morphometry of gametocytes and host cells of Leucocytozoon lovati from the rock ptarmigan (Lagopus mutus) in Japan.

Measurement Minimum–maximum (␮m) MeanϮSD (␮m)

Unparasitized erythrocyte (nϭ60) Length 9.6–12.0 11.0Ϯ0.56 Width 4.8–7.7 6.5Ϯ0.51 Length of nucleus 3.8–6.7 5.0Ϯ0.55 Width of nucleus 1.9–3.9 2.4Ϯ0.37 Macrogametocyte (nϭ63) Length 2.2–27.0 17.6Ϯ4.26 Width 5.9–16.1 10.9Ϯ2.66 Length of nucleus 3.0–8.5 4.8Ϯ1.20 Width of nucleus 1.7–5.1 2.9Ϯ0.84 Length of host cell nucleus 2.2–18.6 13.7Ϯ2.91 Width of host cell nucleus 0.8–11.8 2.9Ϯ2.12 Length of host cell–parasite complex 22.0–60.8 42.7Ϯ8.94 Microgametocyte (nϭ64) Length 15.2–28.7 22.6Ϯ3.17 Width 4.7–15.2 9.1Ϯ2.51 Length of host cell nucleus 8.5–16.6 12.7Ϯ1.72 Width of host cell nucleus 4.7–15.2 9.1Ϯ2.51 Length of host cell–parasite complex 5.6–60.8 45.9Ϯ9.17 length of the gametocytes, host nuclei, and blue and the nucleus had expanded to the the host cells were measured by following periphery farther than the macrogameto- the techniques described by Valkiu¯ nas et cytes. The host cell was remarkably trans- al. (2002). formed due to the infection and several Representative stained blood films from patterns of shape were observed (Fig. 1). infected birds have been deposited in the The microgametocytes were more oval Meguro Parasitological Museum (4-1-1 compared to the macrogametocytes. Both Shimomeguro, Meguro, Tokyo 153-006, ends of long axis of the host cells infected Japan; accession numbers MPM Col. with the micro- and macrogametocytes 18818–18819). were extended. There were many mature Micro- and macrogametocytes of L. lov- gametocytes, but no immature gameto- ati were identified in four (80%) of five cytes were observed. The means and stan- wild birds captured in April and four dard deviations of various measurements (100%) of four wild birds captured in June of the gametocytes and the host cells are (Fig. 1). This is the first report of L. lovati given in Table 1. in grouse in Japan. The captive birds were The measurements of the parasites in not infected. this study were not identical with those The percentage of infected cells per 400 given for L. lovati by Sambon (1908) and leukocytes was 0.32–12.1% (meanϮSD, Fantham (1910), but as pointed out by 2.31Ϯ3.92%) and the Ashford scale varied Bennett et al. (1991) in their redescription from 1 to 3 (meanϮS.D, 1.6Ϯ1.0). No of this species, these parasites are highly mixed infections with other hematozoa pleomorphic. The host cell was greatly were observed. transformed by the parasite in our sam- The macrogametocytes stained a shade ples. Fantham (1910) reported that the ga- of blue and the nucleus stained light pink, metocyte of L. lovati changes its form whereas vacuoles were present in the cy- when growing from immature to mature toplasm. Microgametocytes stained pale stages. The various forms of the gameto- SHORT COMMUNICATIONS 807

cytes observed in this study probably rep- DANO. 2003. Blood parasites in sage-grouse resent different growth stages, although from Nevada and Oregon. Journal of Wildlife Diseases 39: 203–208. more precise studies regarding the possi- FANTHAM, H. B. 1910. Observation on the parasitic bility of mixed infections of more than two protozoa of the red grouse (Lagopus scoticus), species of Leucocytozoon are needed. with a note on the grouse fly. In Proceedings of In Alaska, 144 (90%) of 162 rock ptar- the General Meetings for Scientific Business of migan were infected with L. lovati (Stabler the Zoological Society of London, pp. 692–709. FORBES, M., P. J. WEATHERHEAD, AND G. F. BEN- et al., 1967) and a similarly high preva- NETT. 1994. Blood parasites of blue grouse: Var- lence (89%) of infection was observed iation in prevalence and patterns of interspecific among the birds in this study at Mt. Ta- association. Oecologia 97: 520–525. teyama and Mt. Jiigatake. Our sample size GREINER, E. C. 1991. Leucocytozoonosis in water- was small, however, and additional studies fowl and wild galliform birds. Bulletin of the So- ciety for Vector Ecology 16: 84–93. are needed to determine the various facets HANEDA, K., H. NAKAMURA,A.KOIWAI,T.IIZAWA, of the prevalence of this blood parasite in AND K. TAJIMA. 1985. Distribution and density ptarmigan in Japan. of the rock ptarmigan Lagopus mutus in the The authors are indebted to the mem- southern Japan Alps. Tori. Bulletin of the Orni- bers of Ptarmigan Research Group of To- thological Society of Japan 34: 33–48. [In Japa- nese with English summary.] yama Prefecture and the staff of Ohmachi HOLMSTAD, P. R., A. ANWAR,T.IEZHOVA, AND A. Alpine Museum for sampling at Mt. Ta- SKORPING. 2003. Standard sampling techniques teyama, Mt. Jiigatake, and the Museum. underestimate prevalence of avian hematozoa in willow ptarmigan (Lagopus lagopus). Journal of LITERATURE CITED Wildlife Diseases 39: 354–358. ASHFORD, R. W., I. WYLLIE, AND I. NEWTON. 1990. JORDAN, F. T. W. 1990. Leucocytozoonosis. In Poul- Leucocytozoon toddi in British sparrowhawks Ac- try diseases, F. T. W. Jordan (ed.). 3rd Edition. cipiter nisus: Observation on the dynamics of in- Bailliere Tindall, London, UK, p. 245. fection. Journal of Natural History 24: 1101– SAMBON, L. R. 1908. Remarks on the avian hae- 1107. moprotozoa of the genus Leucocytozoon, Dani- BENNETT, G. F., R. A. EARLE,M.A.PEIRCE,F.W. lewsky. Journal of Tropical Medicine and Hy- HUCHZERMEYER, AND D. SQUIRES-PARSONS. giene 11: 245–248, 325–328. 1991. Avian Leucocytozoidae: The leucocyto- STABLER, R. M., N. J. KITZMILLER, AND R. B. WEE- zoids of the Phasianidae sensu lato. Journal of DEN. 1967. Blood parasites in rock ptarmigan Natural History 25: 1407–1428. from Eagle Summit, Alaska. Journal of Parasitol- ,M.A.PEIRCE, AND R. A. EARLE. 1994. An ogy 53: 1297. annotated checklist of the valid species of Hae- STUHT, J. N., W. W. B OWERMAN, AND D. A. BEST. moproteus, Leucocytozoon (Apicomplexa: Hae- 1999. Leucocytozoonosis in nestling bald eagles mosporida) and Hepatozoon (Apicomplexa: Hae- in Michigan and Minnesota. Journal of Wildlife mogregarinidae). Systematic Parasitology 29: 61– Diseases 35: 608–612. 73. VALKIU¯ NAS, G., T. A. IEZHOVA, AND S. V. MIRONOV. CLARKE, C. H. D. 1935. Blood parasites of ruffed 2002. Leucocytozoon hamiltoni n. sp. (Haemos- grouse (Bonasa umbellus) and spruce grouse porida, Leucocytozoidae) from the Bukharan (Canachites canadensis), with description of Leu- great tit Parus bokharensis. Journal of Parasitol- cocytozoon bonasae n. sp. Canadian Journal of ogy 88: 577–581. Research 12: 646–650. DUNBAR, M. R., S. TORNQUIST, AND M. R. GIOR- Received for publication 23 June 2003.