NOTE Parasitology Plasmodium (Bennettinia) juxtanucleare Infection in a Captive White Eared-Pheasant (Crossoptilon crossoptilon) at a Japanese Zoo
Koichi MURATA1)*, Ryosuke NII1), Emi SASAKI1), Satoshi ISHIKAWA1), Yukita SATO1), Kyoko SAWABE2), Yoshio TSUDA2), Rei MATSUMOTO3), Akemi SUDA3) and Miya UEDA3)
1)College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252–8510, 2)Department of Medical Entomology, National Institute of Infectious Diseases, Toyama 1–23–1, Shinjuku-ku, Tokyo 162–8640 and 3)Yokohama Zoological Garden, 1175–1 Kamishirane-cho, Asahi-ku, Yokohama, Kanagawa 241–0001, Japan
(Received 6 September 2007/Accepted 19 October 2007)
ABSTRACT. An adult male white eared-pheasant (Crossoptilon crossoptilon) at a Japanese zoo exhibited lethargy and emaciation. Micro- scopic examination of a blood smear revealed a haemosporidian parasitic infection. Based on the morphological characteristics and molecular analysis of the parasite, it was identified as Plasmodium (Bennettinia) juxtanucleare. This is the first report of P. juxtanucleare infection in bird species belonging to the genus Crossoptilon. Caution against avian malaria infection is required for the conservation of endangered bird species in zoos. KEY WORDS: haemosporidian parasite, Plasmodium (Bennettinia) juxtanucleare, white eared-pheasant. J. Vet. Med. Sci. 70(2): 203–205, 2008
The white eared-pheasant (Crossoptilon crossoptilon) is propagation program in March 1999. In March 2006, the a Galliformes bird native to the Qinghai, Sichuan, Yunnan bird exhibited lethargy and weakness. No other birds kept and Tibet regions of mainland China, and it is listed as a with or near this individual exhibited the same clinical signs. Near Threatened (NT) species in the 2006 IUCN Red List Since a haemosporidian parasite was detected by blood Category [6]. A captive propagation plan has been designed examination, medical treatment with an antimalarial drug for this species at zoos worldwide, and captive breeding has was started. During the duration of therapy for a year, blood been successful in some zoos. Currently, there are 47 cap- samples were collected 7 times from the brachial wing vein. tive birds worldwide, and 18 birds are present in 3 Japanese Hemacolor® (Merck KGaA, D-64271 Darmstadt, Germany) zoos (International Species Information System; http:// stained blood films were scanned for the presence of the app.isis.org/abstracts/abs.asp). blood parasite with an optical microscope for more than 15 Plasmodium (Bennettinia) juxtanucleare is mainly found min, first at low magnification (× 100) and then at × 1,000 in Phasianidae bird species, including domestic fowl, in the with an oil immersion objective. The WinROOF Profes- widespread zoogeographical regions of the neotropics, Ethi- sional (Mitani Corporation, Fukui, Japan) was used to take opia and the Orient [2, 17]. This parasite was also found to measurements from the digital photomicrographs of the par- infect a variety of wild and captive birds mainly belonging asite. The ratio of infected blood cells was calculated after to the Phasianidae, e.g. the Chinese bamboo partridge (Bam- observing 5,000 blood cells. All results were expressed as busicola thoracica), grey-winged francolin (Francolinus means ± standard deviation of the mean. africanus) and Ceylon jungle fowl (Gallus lafayettei) [7, 13, DNA was extracted from heparinized whole blood sam- 17]. A case of infection in a Corvidae bird and a captive ples at diagnosis and from a frozen blood sample collected black-footed penguin (Spheniscus demersus) were also at arrival by phenol-chloroform method [15]. The nested reported [2, 8]. The effect of the parasitic infection on cap- PCR reaction targeted a partial region of the cytochrome b tive endangered species poses not only individual health genes of Plasmodium was performed in a 25 µl reaction risks but also propagation problems. Here, we present a mixture using primer sets designed by Hellgren et al. [9]. case of P. juxtanucleare infection in a captive white eared- The first and second cycling conditions were as follows: pheasant in order to contribute to the associated veterinary 94°C for 1 min; followed by 30 cycles of 94°C for 1 min, management and the ex situ conservation of this rare spe- 52°C for 1 min, and 72°C for 1 min; and a final extension at cies. 72°C for 5 min. The nested PCR products were directly The affected bird was an 11-year-old male white eared- sequenced from both ends using an ABI PRISMTM 310 pheasant at Yokohama Zoological Garden (35°29’58”N, Genetic Analyzer (Applied Biosystems Japan Ltd., Japan). 139°31’27’E). The bird was hatched at Yokohama Munici- The sequences obtained from this study and those of pal Nogeyama Zoo (35°26’45”N, 139°37’24”E) in June other Plasmodium species in GenBank and DDBJ (Acces- 1995 and was transported to the present facility for a captive sion nos. DQ017964, AB250415, AY099029, AY733090, AJ276844) were aligned using the ClustalW [16], and phy- *CORRESPONDENCE TO: MURATA, K., College of Bioresource Sci- ences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa logenetic analysis was performed using MEGA version 3.1 252–8510, Japan. [12]. Distance analyses were performed using the Kimura e-mail: [email protected] 2-parameter model with the neighbor-joining (NJ) method. 204 K. MURATA ET AL.
Leucocytozoon dubreuli sequence (GenBank Accession no. fied as P. (Bennettinia) juxtanucleare [3, 17, 18]. AY099063) was used as an outgroup to root the tree. Boot- Plasmodium genes were detected in both the samples col- strap analyses used 10,000 replicates. lected at the time of diagnosis and arrival. Alignment of the Examination of the stained blood films revealed that the partial cytochrome b gene of Plasmodium from the white avian malarial parasite of the genus Plasmodium was eared-pheasant resulted in 478 bp in length. The NJ analysis present in the cytoplasm of the red blood cells. During the placed the parasite from this individual in the same cluster clinical period, the extent of parasitemia was 3.7 ± 2.14% as P. juxtanucleare isolated from fowls in Japan [14] and (range: 1.0–7.1%, n=7). No other species of haemosporid- Brazil (cited GenBank), and the percent identity of the para- ian parasites was detected. site from this study with the 478 bp Japanese strain and the Small trophozoites of the parasite were observed adjacent 474 bp Brazilian strain was 100% and 99%, respectively to the erythrocyte nuclei. The gametocytes were round or (Fig. 2). The DDBJ accession number for the partial region oval and small. Most of their size did not exceed the host of cytochrome b is AB302893. cell nucleus, but some gametocytes were lager than the P. juxtanucleare was first described in fowls (Gallus gal- nucleus of its host erythrocyte in size and slightly displaced lus var. domesticus) in Brazil [18]. Although the parasite it to the periphery (Fig. 1). From these morphological char- was found in 6 species from 5 avian orders of Phasianidae acteristics and morphometrics of the parasite, it was identi- after the first report [17], this is the first documentation of P.
Fig. 1. Photos of Plasmodium (Bennettinia) juxtanucleare from the blood film of a captive white eared-pheasant (Crossoptilon cros- soptilon) exhibited lethargy and emaciation. 1: Several trophozoites showing round forms are adjacent to the nucleus of their host erythrocyte. 2: A mature microgametocyte. This clinical stage showed parasitemia level of 3.69%. Bar=10 µm.
Fig. 2. Phylogenetic tree constructed by neighbor joining (NJ) method using the sequences of the partial cytochrome b genes of several Plasmodium juxtanucleare strains, other Plasmodium species and Leucocytozoon dubreuli cited GenBank as the outgroup. Bootstrap analyses involved 10,000 replicates. The percent identity of the P. juxtanucleare from the white eared-pheasant in this study to the 478 bp Japanese strain (AB250415) and the 474 bp Brazilian strain (DQ017964) was 100% and 99%, respectively. PLASMODIUM JUXTANUCLEARE FROM A WHITE EARED-PHEASANT 205 juxtanucleare infection found in a bird of the genus Cros- ence and Technology, Japan and Nihon University Research soptilon. Grants. In the present study, molecular analysis revealed that the partial mitochondrial cytochrome b gene sequences of the REFERENCES parasite detected in the white eared-pheasant were identical to those of P. juxtanucleare isolated from fowls in Japan 1. Al-Dabagh, M.A. 1961. J. Comp. Pathol. 71: 217–221. [14]. Although few informative data are available from the 2. Bennett, G.F., Bishop, M.A. and Peirce, M.A. 1993. Syst. Par- DNA database in order to compare among strains of the par- asitol. 26: 171–179. asite, our molecular analysis data demonstrated that the par- 3. Bennett, G.F. and Warren, M. 1966. J. Parasitol. 52: 565–569. 4. Bennett, G.F., Warren, M. and Cheong, W.H. 1966. J. Parasi- asite belongs not to the Brazilian strain [11] but to the Asian tol. 52: 647–652. strain (Fig. 2). More molecular data is required on the hae- 5. Bennett, G.F. and Warren, M. 1966. J. Parasitol. 52: 653–659. mosporidian parasite and its natural reservoir since it would 6. BirdLife International 2004. IUCN Red List of Threatened provide useful information on the evolution, distribution Species.