Parasitol Res (2008) 103:953957 DOI 10.

1007/s00436-008-1083-4

ORIGINAL PAPER

Two extra chromosomal genomes of Leucocytozoon caulleryi; complete nucleotide sequences of the mitochondrial genome and existence of the apicoplast genome
Sumie Omori & Yukita Sato & Shiho Hirakawa & Takashi Isobe & Masayoshi Yukawa & Koichi Murata

Received: 28 May 2008 / Accepted: 5 June 2008 / Published online: 27 June 2008 # Springer-Verlag 2008

Abstract We analyzed extra chromosomal genomes of avian blood protozoa, Leucocytozoon caulleryi. One of the genomes, the mitochondrial genome was completely sequenced resulting 5,959 bp in length. This genome contained the identical gene organization and contents to that of other avian blood protozoa previously analyzed: three functional genes for cytochrome c oxidase subunit I, III, and cytochrome b with following sets of discontinuous and scrambled 15 ribosomal subunit RNA genes. In addition, the mitochondrial genome was estimated to have the tandem repeated structure as well as previously found in avian Plasmodium species. Furthermore, we found partial gene sequences of apicoplast DNA, another extra chromosomal genome, from L. caulleryi. These sequences were estimated as partial caseinolytic protease C and elongation factor Tu A genes. This report is the first description of two extra chromosomal genomes of L. caulleryi.
S. Omori : Y. Sato (*) : S. Hirakawa : M. Yukawa Laboratory of Biomedical Science, Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, Fujisawa 252-8510, Japan e-mail: sato.yukita@nihon-u.ac.jp T. Isobe National Institute of Animal Health, Tsukuba 305-0856, Japan K. Murata Laboratory of Wildlife Science, Department of Animal Resource Sciences, College of Bioresource Sciences, Nihon University, Fujisawa 252-8510, Japan S. Omori Japan Society for the Promotion of Science (DC1), Tokyo, Japan

Introduction Leucocytozoon caulleryi, an etiologic agent of chicken leucocytozoonosis, is a member of the phylum Apicomplexa (genus Leucocytozoon). This phylum of parasites includes both human pathogens, such as Toxoplasma gondii and Cryptosporidium spp., and economically important chicken pathogen of Eimeria tenella (Joseph et al. 1989). The natural vector of L. caulleryi is the biting midge of Culicoides arakawae; however, other species of Leucocytozoon are transmitted by black flies (Simuliidae; Atkinson and Van-Riper 1991). In Japan, chicken leucocytozoonosis occurs every year and affected to chicken farms with high mortality to the hosts. So, several vaccination methods to L. caulleryi were developed for the prevention of this severely influenced protozoa infection (Isobe et al. 1991; Ito and Gotanda 2004; Ito et al. 2005). Recent progresses of genome studies suggest that several genes unique to the pathogens could be potential targets for drugs or vaccines (Ginsburg et al. 1986; Cai et al. 2003). However, molecular analysis of L. caulleryi is not enough to obtain information for further treatments against infection of this pathogen. Members of the phylum Apicomplexa including genus Leucocytozoon have a unique organelle called apicoplast which has been regarded as an unpigmented chloroplast remnant (Douglas and Penny 1999; Cai et al. 2003). So, like plants, the Apicomplexan protozoa are thought to have two extra chromosomal DNAs; one is found in mitochondria (mt), the other in approximately 35 kb circular molecule resembling algal plastid genome referred to as an apicoplast (Wilson et al. 1994; Douglas 1999). Mt genomes of Plasmodium, representative protozoa of Apicomplexa, have been well studied; however, little is known on the apicoplast genomes of this genus protozoa, especially of avian blood apicomplexa such as Plasmodium spp.,

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Haemoproteus spp., and Leucocytozoon spp. We previously reported the complete nucleotide sequences and structures of the mt genomes of two avian Plasmodium spp., suggesting unique tandem repeat structure of the genomes (Omori et al. 2007). Genetic analysis of another avian blood protozoa of the genus Leucocytozoon have not been enough even on the mt genome. Although partial sequences of the mt cytochrome b (cytb) gene of Leucocytozoon had been used for many phylogenetic analyses (Perkins and Schall 2002; Hellgren et al. 2004; Sato et al. 2007), entire structure of the mt genome is still unknown. Furthermore, on L. caulleryi, no evidences have been reported for existence of the apicoplast genome. Basic information on any genomes should be accumulated for understanding this severely affected infectious agent of L. caulleryi for chicken to prevent infection. In this study, we present two organelle genomes other than chromosomal of L. caulleryi not only for phylogeny but also for further applied studies based on genetics.

Materials and methods L. caulleryi isolated from Gifu, Japan were maintained by serial passages in chickens and were processed for extraction of DNA by using the infected blood. DNA extraction was performed by usual phenol-chloroform procedure (Omori et al. 2007). Primers design and sequencing of the mitochondrial DNA To amplify the mt genome DNA sequences, several oligonucleotide primers were originally designed from conserved regions of the mt DNA of Plasmodium spp. whose complete mt genome sequences have already been deposited to the GenBank database. After obtaining partial sequences of probable mt genome of L. caulleryi, we designed new primers to amplify the rest parts of the genome. Furthermore, we tried another long amplification to obtain all parts of the genome sequences by one polymerase chain reaction (PCR) with several modifications of the above described PCR procedure. PCR conditions, sequencing procedures, and obtained data analyses were referred to our previous study (Omori et al. 2007). Amplification of apicoplast genome sequences We also investigated whether the apicoplast genome exists in L. caulleryi or not. Several oligonucleotide primers were designed from conserved regions of apicoplast-encoded genes, caseinolytic protease C (clpC), and elongation factor Tu A (TufA) in T. gondii, Plasmodium falciparum, and E. tenella whose complete apicoplast genome sequences had already been reported. Several other primer sets previously

used for amplifying apicoplast genes of avian blood protozoa (Rathore et al. 2001; Perkins et al. 2007; Martinsen et al. 2008) were also applied in this study. PCR was generally carried out as described previously (Omori et al. 2007), applying GXL Taq DNA-polymerase (TaKaRa) for more effective amplification. Condition of PCR by using GXL Taq DNA-polymerase as below: in a 25 l reaction volume containing 10Ex Taq Buffer (Mg2+ free), 0.4 mM dNTPs, 0.4 M of the two primers, 4 mM MgCl 2 , 2.5 U of Ex Taq DNA-polymerase (TaKaRa), and 2 l of template DNA solution and/or in a same volume containing 5GXL Taq Buffer (Mg2+), 0.4 mM dNTPs, 0.4 M of the two primers, 2.5 U of GXL Taq DNA-polymerase (TaKaRa), and 1 l of template DNA solution. The condition of the thermoprofile was followed: initial 2 min of denaturation at 94C was followed by 50 cycles consisting of 30 s at 94C (denaturation), 30 s at 65C (annealing), and 5 min at 72C (elongation). The reaction was finalized by another 10 min at 72C. Electrophoresis and consequent sequencing were implemented as described previously (Omori et al. 2007). Obtained sequences were assembled for comparing similarity to known apicoplast genes in GenBank.

Results Mitochondrial genome Putative mt genome size of L. caulleryi obtained by our PCR procedure was estimated to be 5,959 bp. Constructed long sequences by joining contiguous PCR amplicons are identical to those fragments obtained by one long PCR reaction. GC contents of the genome were 31.2%. This high AT content is similar to other avian blood protozoa, such as Plasmodium gallinaceum with 31.3% of GC ratio (Omori et al. 2007). The complete mt genome of L. caulleryi was deposited to the GenBank database and was issued the accession number, AB302215. Probable gene organization map of the mt genome of L. caulleryi is shown in Fig. 1. Three functional genes of cytochrome c oxidase subunit I (COI), III (COIII), and cytb were estimated with 15 rRNA gene pieces. Only the putative direction of translation for COIII gene was opposite to other two protein-coding genes. Tandem repeated genome unit structures were also found in L. caulleryi as well as those identified in P. gallinaceum and Plasmodium juxtanucleare (Omori et al. 2007). Apicoplast genome Obtained sequences from L. caulleryi DNA by amplifying possible two apicoplast genes of clpC and TufA were firstly

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Fig. 1 Putative gene organization map of the mitochondrial genomes of avian blood protozoa, Leucocytozoon caulleryi. The gene positions and numbers attached with large and small subunit ribosomal RNA units were inferred from previous transcriptional studies of P.

gallinaceum mitochondrial DNA. Arrows indicate the direction of possible transcription. Fragmented and scrambled large subunit and small subunit rRNA genes are numbered in ascending order as they would appear 5 to 3 in conventional rRNA

inquired to homology search by using Basic Local Alignment Search Tool, showing higher similarities to any apicoplast genes than others. The nucleotide sequences of each gene of L. caulleryi, P. gallinaceum, and P. juxtanucleare were deposited to the GenBank database and were issued accession numbers (L. caulleryiclpC: AB435376, TufA: AB435379, P. gallinaceumclpC: AB435377, TufA: AB435380, P. juxtanucleareclpC: AB435378, TufA: AB435381, respectively). Confirmed sequences were also aligned with the same genes of three known avian malaria protozoa, P. relictum, Haemoproteus syrnii, and Haemoproteus turtur, one rodent malaria protozoa, Plasmodium yoelii, one human malaria protozoa, P. falciparum, and the other apicomplexa protozoa, E. tenella and T. gondii. Similarities among eight apicomplexa species were shown

in Table 1. The nucleotide sequences of each gene of L. caulleryi showed closer homology to avian malaria protozoa than mammalian malaria protozoa, E. tenella and T. gondii.

Discussion In this study, we suggested that L. caulleryi has two extra chromosomal genomes as found in other apicomplexa protozoa for the first time; we revealed the complete DNA sequences and the structure of the mt genome and even suggested the presence of an apicoplast genome. We also found that the mt genome of L. caulleryi contained repeated tandem structure as well as avian Plasmodium for

Table 1 Comparison of sequence homology by using partial length of each gene (clpC and TufA) in nucleotide among nine Apicomplexa species P. gallinaceum* L. caulleryi * P. gallinaceum* P. relictum H. syrnii H. turtur P. yoelii P. falciparum T. gondii 86.5 86.8 P. relictum 85.7 ND 88.9 ND H. syrnii 84.6 ND 85.7 ND 85.9 ND H. turtur 84.2 ND 86.1 ND 84.6 ND 94.1 ND P. yoelii 81.8 84.7 85.7 87.2 85.1 ND 84.6 ND 84.4 ND P. falciparum 85.3 84.5 89.5 90.6 87.5 ND 88.3 ND 86.9 ND 87.3 88.7 T. gondii 70.9 71.2 69.1 71.1 70.7 ND 68.3 ND 66.5 ND 67.7 70.3 69.3 71.1 E. tenella 68.5 70.5 67.1 73.0 68.7 ND 66.9 ND 65.9 ND 67.3 69.3 68.5 71.5 72.4 76.2

Numbers indicate each percent of homology of the two genes (upper: clpC, 504 bp and lower: TufA, 672 bp) in nucleotide. *Samples studied in the present report. ND Data are not shown in GenBank database and this research.

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Parasitol Res (2008) 103:953957 University Research Grants, and a Grant-in-Aid for JSPS Fellows (No. 203886) from the Japan Society for the Promotion of Science.

the first time. This tandem repeat of the mt genome may be common feature among apicomplexa protozoa. Several studies revealed the partial mt cytb gene and/or apicoplast genes of Leucocytozoon for phylogenetic investigations (Hellgren et al. 2004;Martinsen et al. 2008); however, understanding of the whole genome structures seems to be important in that finding more adequate sites for phylogenetic comparison and probable antiprotozoa drugs derived from proteins of L. caulleryi. In fact, the mt genome of human malaria protozoa has been supposed to be one of the potential antimalarial drug targets (Ginsburg et al. 1986). The apicoplast is essential and specific to the parasites for the growth, and their animal hosts do not possess such a plant type organelle so it is possible that this organelle may provide novel targets for drugs and vaccines against L. caulleryi infection. Several trials for vaccination and/or inducing immunity to L. caulleryi infection in chicken have been reported in Japan (Isobe et al. 1991; Ito and Gotanda 2004; Ito et al. 2005). L. caulleryi infection causes significant economic losses in poultry industries among the Asian countries (Morii et al. 1981; Yu and Wang 2001). Present prevention procedures are mainly vaccination-based on above-mentioned studies; however, if resistant strains of L. caulleryi occur to those immunological preventions, our findings could serve as possible new preventative measurements because apicoplast-specific proteins were thought to block the growth of the Apicomplexa (Waller et al. 1998; Surolia and Surolia 2001). A previous study showed that three phylogenetic trees obtained from analysis of independent genome genes, plastid clpC, mt cytb, and nuclear small subunit ribosomal RNA of eight Plasmodium species, resulted conflicting topologies each other (Rathore et al. 2001). It could be suggested that if the genes have different rates and patterns of evolution, such analysis based on single gene phylogenies may induce many poorly supported nodes in the trees. However, a simultaneous three genomes phylogeny of malaria parasites combining nuclear and two organelle genes suggested a topology with highly supported nodes (Martinsen et al. 2008). So, further genetic analysis of L. caulleryi is possible by using our results of the complete mitochondrial and partial apicoplast genome sequences. Detailed analysis of L. caulleryi even adding biological characteristics such as morphology, life cycle, and hostparasite relationship could reveal the phylogenetic status of this severe pathogenic Leucocytozoon species among blood parasites of birds.
Acknowledgements The authors are very grateful for Motohashi A. for her technical assistances. This study was partially supported by the Academic Frontier Project Surveillance and control for zoonoses and High-Tech Research Center Project for Private Universities matching funds subsidy from Ministry of Education, Culture, Sports, Science and Technology of Japan, Global Environment Research Fund of the Ministry of the Environment of Japan (F-062), Nihon

References
Atkinson CT, Van-Riper C (1991) Pathogenicity and epizootiology of avian haematozoa: Plasmodium, Leucocytozoon and Haemoproteus. In: Loye JE, Zuk M (eds) Bird-parasite interactions: ecology, evolution and behaviour. University of Oxford Press, Oxford, pp 1948 Cai X, Fuller AL, McDougald LR, Zhu G (2003) Apicoplast genome of the coccidian Eimeria tenella. Gene 321:3946 Douglas SE (1999) Evolutionary history of plastids. Biol Bull 196:397399 Douglas SE, Penny SL (1999) The plastid genome of the cryptophyte alga, Guillardia theta: complete sequence and conserved synteny groups confirm its common ancestry with red algae. J Mol Evol 48:236244 Ginsburg H, Divo AA, Geary TG, Boland MT, Jensen JB (1986) Effects of mitochondrial inhibitors on intraerythrocytic Plasmodium falciparum in in vitro cultures. J Protozool 33:121125 Hellgren O, Waldenstrom J, Bensch S (2004) A new PCR assay for simultaneous studies of Leucocytozoon, Plasmodium, and Haemoproteus from avian blood. J Parasitol 90:797802 Isobe T, Suzuki K, Yoshihara S (1991) Protection against chicken leucocytozoonosis provided by immunization with spleen homogenate infected with Leucocytozoon caulleryi. Avian Dis 35:559562 Ito A, Gotanda T (2004) Field efficacy of recombinant R7 vaccine against chicken Leucocytozoonosis. J Vet Med Sci 66:483487 Ito A, Gotanda T, Kobayashi S, Kume K, Sugimoto C, Matsumura T (2005) Increase of antibody titer against Leucocytozoon caulleryi by oral administration of recombinant R7 antigen. J Vet Med Sci 67:211213 Joseph JT, Aldritt SM, Unnasch T, Puijalon O, Wirth DF (1989) Characterization of a conserved extrachromosomal element isolated from the avian malarial parasite Plasmodium gallinaceum. Mol Cell Biol 9:36213629 Martinsen ES, Perkins SL, Schall JJ (2008) A three-genome phylogeny of malaria parasites (Plasmodium and closely related genera): Evolution of life-history traits and host switches. Mol Phylogenet Evol 47:261273 Morii T, Shiihara T, Lee YC, Manuel MF, Nakamura K, Iijima T, Hoji K (1981) Seroimmunological and parasitological surveys of Leucocytozoon caulleryi infection in chickens in several Asian countries. Int J Parasitol 11:187190 Omori S, Sato Y, Isobe T, Yukawa M, Murata K (2007) Complete nucleotide sequences of the mitochondrial genomes of two avian malaria protozoa, Plasmodium gallinaceum and Plasmodium juxtanucleare. Parasitol Res 100:661664 Perkins SL, Schall JJ (2002) A molecular phylogeny of malarial parasites recovered from cytochrome b gene sequences. J Parasitol 88:972978 Perkins SL, Sarkar IN, Carter R (2007) The phylogeny of rodent malaria parasites: simultaneous analysis across three genomes. Infect Genet Evol 7:7483 Rathore D, Wahl AM, Sullivan M, McCutchan TF (2001) A phylogenetic comparison of gene trees constructed from plastid, mitochondrial and genomic DNA of Plasmodium species. Mol Biochem Parasitol 114:8994 Sato Y, Hagihara M, Yamaguchi T, Yukawa M, Murata K (2007) Phylogenetic comparison of Leucocytozoon spp. from wild birds of Japan. J Vet Med Sci 69:5559

Parasitol Res (2008) 103:953957 Surolia N, Surolia A (2001) Triclosan offers protection against blood stages of malaria by inhibiting enoyl-ACP reductase of Plasmodium falciparum. Nat Med 7:167173 Waller RF, Keeling PJ, Donald RG, Striepen B, Handman E, Lang-Unnasch N, Cowman AF, Besra GS, Roos DS, McFadden GI (1998) Nuclearencoded proteins target to the plastid in Toxoplasma gondii and Plasmodium falciparum. Proc Natl Acad Sci U S A 95:1235212357

957 Wilson RJ, Williamson DH, Preiser P (1994) Malaria and other Apicomplexans: the plant connection. Infect Agents Dis 3:29 37 Yu CY, Wang JS (2001) Role of chicken serum in inhibiting Leucocytozoon caulleryi development in Culicoides arakawae infected by membrane-feeding of infective blood meals. Parasitol Res 87:698701