FULL PAPER Parasitology

Prevalence of Blood Parasites in Japanese Wild

Koichi MURATA1)

1)Department of Wildlife Science, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252–8510, Japan

(Received 12 October 2001/Accepted 7 May 2002)

ABSTRACT. The prevalence of blood parasites was investigated in 701 Japanese wild birds for 13 years from January, 1988 to March, 2001. Most of the injured or sick birds were caught in the suburbs of Kobe City, Hyogo Prefecture and brought to the zoo for clinical care. Among all the birds examined, 10.6% were infected with hematozoa belonging to three genera as Plasmodium (1.7% of the samples), Haemoproteus (5.1% of the samples) and Leucocytozoon (4.6% of the samples), and two birds (0.29% of the samples), a Japanese gros- beak (Coccothraustes personatus) and a dusky thrush (Turdus naumanni), were infected with microfilariae. Mixed infection with Leu- cocytozoon sp. and Haemoproteus sp. was observed in 6 individuals of 4 species and that with Leucocytozoon sp. and microfilariae was observed in 2 individuals of 2 species of . Relatively high positive rates were 75%(3/4) in the scops owl (Otus scops), 71.4% (10/ 14) in the ural owl (Strix uralensis), 57.7% (15/26) in the jungle crow (Corvus macrorhynchos), 57.1% (4/7) in the black-tailed gull (Larus crassirostris), 55.6% (5/9) in the brown hawk owl (Ninox scutulata), 41% (16/39) in the carrion crow (Corvus corone) and 24.1% (7/29) in the night heron (Nycticorax nicticorax). KEY WORDS: Haemoproteus, Japanese wild bird, Leucocytozoon, microfilaria, Plasmodium. J. Vet. Med. Sci. 64(9): 785–790, 2002

Four genera, Plasmodium, Haemoproteus, Leucocyto- be a good source to research the spread of avian blood para- zoon and Trypanosoma, of blood parasite are well known as sites in natural populations of birds in Japan. The informa- avian hematozoa observed in a variety of bird species. tion could be useful for the veterinary medical care of Microfilariae were also detected from wild birds in the captive exotic birds but also of wild birds. The author pre- blood [2, 9, 18]. Blood sucking arthropods such as Culex viously reported the prevalence of hematozoa from 69 Japa- spp. and Aedes spp. play an important role as the vectors of nese wild birds brought to the zoo between 1988 and 1989 these parasites [2]. Recently, a number of scientific studies [15]. In the present study, the informations, including the have been published worldwide about avian blood parasites. former records of avian blood parasites in Japan, were addi- They were carried out not only from the biological interests, tionally examined. such as , but also for the conservation of endan- gered avian species by controlling the blood parasites [3–7, MATERIALS AND METHODS 18]. The exact cases where wild bird species declined and A total of 701 Japanese wild birds belonging to 69 species extinct by the infection with blood parasites and their vector in 32 families of 10 orders were surveyed for 13 years from are not a few. In Hawaii, the mosquitoes introduced into the January, 1988 to March, 2001. The birds were caught island transmit P. relictum to the Amakihi (Hernignathus mainly in the suburbs of Kobe, Hyogo Prefecture situated at virens) and Hawaiian crow (Corvus hawaiiensis), and the approximately 34°N; 135°W, except for several species of bird species are declining in population [4, 25]. Leucocyto- birds caught outside the city, and transported to the veteri- zoon spp. occurred in some endemic species of bird in the nary hospital at Kobe Municipal Oji Zoo for clinical care. Mascarene Islands [18]. The birds of which population is Most birds were debilitated or injured at the time of arrival. limited in an island can easily receive catastrophic damage The blood samples from 12 alpine accentors (Prunella col- by hematozoa transmitted by arthropod vectors introduced laris) were supplied by Dr. Nakamura in 1989. The birds into the island [4, 9, 20, 25]. Similarly, it becomes indis- were caught around Mt. Norikura situated at approximately pensable to pay attention to blood parasites as well as patho- 36°N; 137°W, about 2,800 m elevation, in Nagano Prefec- genic bacteria, rickettsia and viruses, when biologists or ture for scientific study under the permission of Environ- veterinarians are planning conservation of endangered bird mental Agency of the Japanese Government. species. The birds species examined are listed in Tables 1–1 to 1– In Japan, recent information regarding blood parasite 3 according to the previous report which showed the molec- infection in wild birds is inadequate compared with that in ular biological classification by the DNA-DNA hybridiza- other countries, except for some studies concerning the clas- tion technique [23]. sification and biology of avian hematozoa [10, 16, 22]. Blood samples were collected from the birds at the wing Many wild birds were caught in the suburbs of town because vein using a heparinized syringe. Blood smears were made of injury or diseases with some clinical signs and brought to and fixed with methanol immediately after blood collection. zoos for medical care every year. These rescued birds will Prepared blood smears were stained with Giemsa solution or 786 K. MURATA

Table 1-1. Blood parasites found in Japanese wild birds

Bird species Number of birds Number of birds infected Parasitea) Number of birds with Order Family Species examined with parasites H P L mf mixed infection Galliformes Phasianidae Symaticus soemmerringi 10 Phasianus versicolor 20 Anseriformes Anatidae Anas strepera 30 Anas crecca 10 Anas poecilorhyncha 60 Aythya ferina 511 Piciformes Picidae Picoides major 10 Coraciiformes Coraciidae Halcyon coromanda 10 Cuculiformes Cuculidae Cuculus poliocephalus 20 Strigiformes Strigidae Otus scops 42211 (H/L) Otus bakkamoena 111 Ninox scultulata 955 Strix uralensis 14 9 9 1 1 (H/L) Asio flammeus 20 Caprimulgidae Caprimulgus indicus 10 Columbiformes Columbidae Columba livia var. domestica 278 0 Streptopelia orientalis 71 1 1 Treron sieboldii 61 1 Gruiformes Gruidae Fulica atra 10

Table 1-2. Blood parasites found in Japanese wild birds

Bird species Number of birds Number of birds infected Parasitea) Number of birds with Order Family Species examined with parasites H P L mf mixed infection Ciconiiformes Scolopacidae Heteroscelus brevipes 10 Phalaropus lobatus 10 Scolopax rusticola 71 1 Gallinago gallinago 11 1 Charadriidae Charadrius alexandrinus 10 Laridae Larus crassirostris 744 Larus agrentatus 20 Larus ridibundus 50 Rissa tridactyla 10 Sterna fuscata 10 Sterna albifrons 10 Synthliboramphus antiquus 20 Accipitridae Accipiter nisus 30 Accipiter gentilis 20 Milvus migrans 12 0 Falconidae Falco peregrinus 20 Podicipedidae Tachybaptus ruficollis 10 Ardeidae Ardea cinerea 17 0 Ardeidae Egretta alba 40 Egretta garzetta 30 Egretta intermedia 10 Bubulcus ibis 20 Nycticorax nicticorax 29 7 3 4 Ixobrychus sinensis 311 Butorides striatus 50 Procellariidae Pterodroma hypoleuca 10 Calonectris leucomelas 60 Hydorbatidae Oceanodroma monorhis 10

Hemacolor® (Merck Co.), and each slide was examined identification of the parasites to the genus level. Briefly, using a microscope with two magnifications (× 200 and Plasmodium sp. was identified by the existence of trophozo- × 400) at least 200 different fields. High magnification ites and gametocytes usually with a round or elongate shape under oil immersion (× 1,000) was used for morphological in the red blood cells (Fig. 1-A). The gametocytes of Hea- BLOOD PARASITES OF JAPANESE WILD BIRDS 787

Table 1-3. Blood parasites found in Japanese wild birds

Bird species Number of birds Number of birds infected Parasitea) Number of birds with Order Family Species examined with parasites H P L mf mixed infection Passeriformes Laniidae Lanius bucephalus 20 Corvidae Garrulus glandarius 10 Corvus corone 39 16 4 14 2 (H/L) Corvus macrorhynchos 26 15 3 4 10 2 (H/L) Bombycillidae Bombycilla japonica 10 Muscicapidae Zoothera dauma 30 Turdus cardis 10 Turdus chrysolaus 10 Turdus pallidus 30 Turdus naumanni 41 111 (L/mf) Cettia squameiceps 10 Sturnidae Sturnus cineraceus 15 1 1 Paridae Parus major 10 Hirundinidae Hirundo rustica 40 Hirundo daurica 30 Pycnonotidae Hyspsipetes amaurotis 33 5 1 2 2 Zosteropidae Zosterops japonicus 111 Alaudidae Alauda arvensis 10 Fringillidae Coccothraustes personatus 31 111 (L/mf) Coccothraustes coccothnaustes10 Prunellidae Prunella collaris 12 0 Passeridae Passer montanus 14 0 Total 701 74 36 12 32 2 8 a) The symbols used in the table means as follows: H: Haemoproteus sp., P: Plasmodium sp., L: Leucocytozoon sp. and mf: Microfilaria. moproteus sp. were spherical, elongate or banana-shaped of the ural owl (Strix uralensis), 65.4% (17/26) of the jungle with pigment granules encircling the nucleus of host eryth- crow (Corvus macrorhynchos), 57.1% (4/7) of the black- rocyte (Fig. 1-B). Leucocytozoon sp. was identified by the tailed gull (Larus crassirostris), 55.6% (5/9) of the brown dark stained cytoplasm with a round or elongate form filling hawk owl (Ninox scutulata), 46.1% (18/39) of the carrion the host cells (Fig. 1-C). crow (Corvus corone) and 24.1% (7/29) of the night heron (Nycticorax nicticorax). In the birds belonging to the order RESULTS Strigiformes, 63.3% (19/30) were infected with Haemopro- teus sp. and/or Leucocytozoon sp.. From 278 blood samples In this survey, hematozoa belonging to the following of the domestic pigeon (Columba livia var. domestica) of three genera were detected; Plasmodium spp., Haemopro- the order Columbiformes, no hematozoa were detected. On teus spp. and Leucocytozoon spp. (Tables 1–1 to 1–3). In the other hand, Haemoproteus sp. was present in 16.7% of 6 total, the hematozoa were present in 10.6% of 701 individu- blood samples from the Japanese green pigeon (Treron sie- als belonging to Anseriformes, Strigiformes, Columbi- boldii) belonging to the same order. No hematozoa infec- formes, Ciconiiformes and Passeriformes. The positive tion was observed in 12 alpine accentors (Table 1–3). birds were detected in 27.5% of 69 species, 37.5% of 32 Microfilariae in the dusky thrush (Fig. 1-D) were first families and 50% of 10 orders. Regarding each species of recorded from the natural host in Japan. hematozoa, Plasmodium spp., Haemoproteus spp. and Leu- Two serious clinical cases caused by heavy infection with cocytozoon spp. were present in 1.7%, 5.1% and 4.6% of the Leucocytozoon sp. or Plasmodium sp. were found in the car- samples, respectively. Mixed infections with Leucocyto- rion crow. zoon sp. and Haemoproteus sp. were observed in 6 birds belonging to 4 species, and those with Leucocytozoon sp. DISCUSSION and microfilaria were in 2 birds belonging to 2 species (Tables 1–1 to 1–3). Microfilariae were found in the blood This study demonstrated the recent information about the smears from a Japanese grosbeak (Coccothraustes per- prevalence of blood parasites from Japanese wild birds. The sonatus) and a dusky thrush (Turdus naumanni) (Table 1–3, previous study by Ogawa [16] showed that hematozoa were Fig.1-D). The annual difference of prevalence rate from present in only 1% of 1,478 Japanese birds belonging to 54 1988 to 2001 was not significant. species. The positive rate (10.6%) in the present study was The relatively high prevalence of hematozoa was found significantly higher than his findings. However, McClure et in 75% (3/4) of the scops owl (Otus scops), 71.4% (10/14) al. [13] reported 16.3% of 9,026 smears from wild birds in 788 K. MURATA

Fig. 1. Blood parasites from Japanese wild birds. A: a gametocyte (arrow) of Haemoproteus sp. from a scops owl (Otus scops). Bar=13 µm, B: several trophozoites of Plasmodium sp. (arrows) detected form a jungle crow (Corvus corone). Bar=16 µm, C: macrogameto- cyte (arrow) and microgametocyte (double arrows) of Leucocytozoon sp. from a Japanese grosbeak (Coccothraustes personatus). Bar=12 µm, D: four microfilariae from a dusky thrush (Turdus naumanni). Bar=80 µm. the Asian tropical sub-region were positive for hematozoa species of wild birds [8, 10, 16, 22]. Most of them are Pas- [11, 12]. Kano and Kimura [10] found several species of seriformes such as the (Coccothraustes cocco- blood parasites in 22.9% of 310 wild birds from Eastern thraustes), siskin (Carduelis spinus), long-tailed rose Japan. Considering these two reports, the number of posi- (Uragus sibiricus), brambling (Fringilla montifringilla), tive cases shown in the present survey appears to reflect the bull-headed shrike (Lanius bucephalus), Japanese proper state of blood parasites infection among Japanese (Bombycilla japonica) and jungle crow. The hematozoa wild birds. were also detected from the collared scops owl (Otus bakka- A variety of Japanese wild birds were indicated as the moena), scops owl (Otus scops) and ural owl (Scops uralen- hosts of blood parasites [10, 16]. The grey starling and the sis) belonging to Strigiformes [10, 16]. Sakamoto et al. [22] jungle crow were newly added as the natural hosts of Hae- and Hayashi et al. [8] reported the high occurrence of Try- moproteus sp. and Plasmodium sp., respectively in the pre- panosoma sp. in crows at Kagoshima and Tottori districts. vious investigation [15]. In addition, the microfilaria The reason why no Trypanosoma sp. was detected from 65 infection in the dusky thrush was first reported in this sur- crows in the present survey might be due to the difference in vey. If similar surveys are made on various species of bird sampling region or season. A survey over a wider range over a wide range of Japan, various types of parasitism may through several seasons would be necessary to clarify the be observed. spread of the hematozoa in crows of Japan. In addition, a No blood parasite was detected from the alpine accentors more precise study should be carried out on the age-, sex- captured at the highlands. It is assumed that the possibility and season-related prevalence of each blood parasite of infection with blood parasites is rare in the habitat where between selected species of bird. the arthropod vectors infrequently appear [4, 5]. By investi- The present survey suggested that the prevalence of gating blood parasites in some bird species living at high- microfilaria infection in Japanese native birds appears to be lands such as the rock ptarmigan (Logopus mutus), the low such as 0.29%. Microfilariae have been detected from relation between the vertical distribution of birds and their the hawfinch, Japanese grosbeak, grey bunting (Emberiza parasite infection will be clarified. It is also important to variabilis), Siberian meadow bunting (E. cioides) and crow survey and monitor whether an arthropod vector of avian in Japan [8, 10]. The dusky thrush was added to the above- blood parasites could gradually expand its habitat to a high mentioned species as a natural host of microfilaria. It is altitude along with the global warming in recent years. assumed that microfilariae are non-pathogenic [19, 21]. In Japan, Trypanosoma spp. were detected from several Despite a histopathological survey of some birds BLOOD PARASITES OF JAPANESE WILD BIRDS 789 infected with numerous microfilariae, no cellular reaction ACKNOWLEDGEMENTS. I express great thanks to Dr. was observed to the parasites [24]. Regarding the low prev- Akira Miyata of Oita Medical College who gave useful alence and low pathogenicity, the infection with microfilar- advice and information of hematozoa in Japanese wild iae without adults may produce no harmful effect in wild birds, Dr. Marshall Laird who provided many scientific bird populations. papers on avian malaria in the Asian region and Dr. Masa- The influence of parasitic infection upon the sex selection hiko Nakamura of Joetsu University of Education who sup- has been an issue in the field of sociobiology [9, 19]. Patho- plied the blood samples of the alpine accentor. genicity of blood parasites, however, is not well understood although parasites are detected from many species of wild REFERENCES birds [16, 21]. To offer a good solution to the subject, it is necessary to investigate the blood parasite and its pathoge- 1. Beier, J.C. and Stoskopf, M.K. 1980. The epidemiology of nicity in various bird species. However, such caution is not avian malaria in black-footed penguins Spheniscus demersus. needed for Haemoproteus spp. in some bird species because J. Zoo Anim. Med. 11: 99–105. the pathogenicity of the hematozoa is thought to be low 2. Bennett, G.F. 1987. Hematozoa, pp. 120–128. In: Companion Bird Medicine (Burr, E.W. ed.), The Iowa State University [17]. At least for birds belonging to Strigiformes, the infec- Press, Ames, Iowa. tion with Haemoproteus spp. may not be so important unless 3. Deviche, P., Greiner, E.C. and Manteca, X. 2001. Interspecific the birds fall into immune deficiency [12], because owls variability of prevalence in blood parasites of adult passerine without any clinical signs developed parasitemia with the birds during the breeding season in Alaska. J. Wildl. Dis. 37: hematozoa (54.8%: 17/31) in the present study. 28–35. Hematozoa infection has been suggested to be species- 4. Feldman, R.A., Freed, L.A. and Cann, R.L. 1995. A PCR test specific, but a fatal case was reported on interspecific infec- for avian malaria in Hawaiian birds. Mol. Ecol. 4: 663–673. tion in Passeriformes [10]. The author previously reported 5. Fix, A.S., Waterhouse, C., Greiner, E.C. and Stoskkopf, M.K. that a carrion crow inoculated with Plasmodium sp. from a 1988. Plasmodium relictum as cause of avian malaria in wild- caught magellanic penguins (Spheniscus magellanicus). J. jungle crow became infected and died of acute parasitemia Wildl. Dis. 24: 610–619. 16 days post-inoculation [15]. This suggests that special 6. Fukui, D., Murata, K., Bando, G., Kosuge, M. and Yamaguchi, caution is needed for preventing the spread of avian malaria M. 2002. Triple infection with avian haematozoa (Haemopro- from its natural hosts to non-natural hosts. In the present teus sp., Leucocytozoon sp., Trypanosoma sp.) in imported study, 1.7% of 701 birds were infected with Plasmodium boreal owls (Aegolius funereus). J. Hokkaido Vet. Med. Assoc. spp. and they occurred in 24.1% of 29 night herons that 46: 10–12 (in Japanese). occasionally form a colony in and around zoo facilities. 7. Hatchwell, B.J., Wood, M.J., Anwar, M.A., Chamberlain, D.E. This may be a problem in the captive breeding program of and Perrinns, C.M. 2001. The hematozoan parasites of com- the endangered Ciconiiformes birds such as the Japanese mon blackbirds Turdus merula: associations with host condi- crested ibis (Nipponia nippon) and Oriental white stork tion. Ibis 143: 420–426. (Ciconia boyciana), and also of penguins which were 8. Hayashi, T., Morita, M., Ouchi, M., Norimura, R., Suzuki, S., Nakamura, M. and Fujii, T. 1998. A hemosite survey of wild recently re-classified into the same order as the heron by crows in Japan. Bull. Fac. Agric. Tottori Univ. 51: 131–136. DNA analysis [23]. Actually, P. relictum and P. elongatum 9. Jenkins, C.D., Temple, S.A., Riper, C.V. and Hansen, W.R. maintain the cycle between wild birds and mosquitoes and 1989. Disease-related aspects of conserving the endangered has been known to be a causative agent of mortality in cap- Hawaiian crow. pp. 77–87. In: Diseases and Threatened Birds tive penguins in Europe and North America [1, 5, 18]. (Cooper, J.E. ed.), ICBP, England. Little attention has been paid to the infection with blood 10. Kano, R. and Kimura, M. 1950. Natural infection of blood par- parasites at zoos and aquariums in Japan, and the diseases asites in Japanese wild birds. Jpn. J. Bact. 5: 103–104. caused by blood parasites tend to be overlooked at quaran- 11. Laird, M. 1997. Avian Malaria in the Asian Tropical Subre- tine. However, some studies have been made on hematozoa gion, Springer, Singapore. infection in imported exotic birds such as the sulphur- 12. Markus, M.B. and Oosthuizen, J.H. 1972. Pathogenicity of Haemoproteus columbae. Trans. R. Soc. Trop. Med. Hyg. 66: crested cockatoo (Cacatua sulphurea), salmon-crested 186–187. cockatoo (C. moluccensis) and boreal owl (Aegolius 13. McClure, H.E., Poonswas, P., Greiner, E.C. and Laird, M. funereus) [6, 14]. It is necessary to recognize blood para- 1978. Haematozoa in the Birds of Eastern and Southern Asia, sites as important etiologic agents and give attention to Memorial University of Newfoundland, Canada. infections transmitted from imported exotic birds to captive 14. Murata, K. 1990. Avian haematozoa and microfilaria infec- or wild birds. If native species of birds are naive to an tions of imported psittacine birds. J. Jpn. Vet. Med. Assoc. 43: imported blood parasite, catastrophic losses may occur 271–274 (in Japanese with English summary). among them. It is, therefore, necessary to give serious atten- 15. Murata, K. 1990. A survey of haematozoa infection in Japa- tion to blood parasites including the vector control and to nese wild birds. J. Jpn. Assoc. Zool. Aqua. 32: 85–89 (in Japa- monitor the spread of the parasites in wild bird populations nese with English summary). 16. Ogawa, K. 1911. Notizen über die blutparasitischen Protozoen from the aspect of veterinary wildlife management. This is bei japanischen Vögeln. Arch. Protist. Jena. 24: 119–126. especially important for island nations such as Japan. 17. Peirce, M.A. 1969. Blood parasites found in imported birds at post-mortem examination. Vet. Rec. 84: 113–116. 790 K. MURATA

18. Peirce, M.A. 1989. The significance of avian haematozoa in S. 1981. I. Parasites of Corbus macrorhynchos in Kagoshima conservation strategies, pp. 69–76. In: Diseases and Threat- District. Bull. Fac. Agric., Kagoshima Univ. 31: 83–93. ened Birds (Cooper, J.E. ed.), ICBP, England. 23. Sibley, C.G. and Monroe, B.L. 1990. Distribution and Taxon- 19. Reppas, G.P., Hartley, W.J. and Gill, P.A. 1995. Microfila- omy of Birds of the World. Yale University Press, New Haven. raemia in Australian native birds. Aust. Vet. J. 72: 356–357. 24. Tsai, S.S., Hirai, K. and Itakura, C. 1992. Histopathological 20. Riper, C.V., Riper, S.G.V., Goff, M.L. and Laird, M. 1986. survey of protozoa, helminths and acarids of imported and The epizootiology and ecological significance of malaria on local psittacine and passerine birds in Japan. Jpn. J. Vet. Res. the birds of Hawaii. Ecol. Monog. 56: 327–344. 40: 161–174. 21. Ritchie, B.W., Harrison, G.J. and Harrison, L.R. 1994. Avian 25. Warner, R.E. 1968. The role of introduced diseases in the Medicine: Principles and Application, Winger Publishing, Inc., of the endemic Hawaiian avifauna. Condor 70: 101– Florida. 120. 22. Sakamoto, T., Kono, I., Yasuda, N., Sakoh, T. and Kawabata,