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The Fine Structure of the Merozoites and Gametocytes of Leucocytozoon caulleryi
Zbl. Bakt. Bakt. Hyg., I. Abt. Orig. A 250, 198-205 (1981) (1981) Zbl.
Department of Parasitology, School of Medicine, Kyorin University, Mitaka, Tokyo 181, Japan
The Fine Structure of the Merozoites and Gametocytes of Leucocytozoon caulleryi * UJIMA, and TSUTOMU MORU, TOSHIHIRO MATSUI, TOSHIHIKO UlIMA, MINORU FUKUDA
Figures· Received February 10, 1981 With 6 Figures' 1981
Summary The fine structure of the merozoites and and gametocytes of Leucocytozoon caulleryi from from The the peripheral blood of chickens was studied by means of electron microscopy. Merozoites and are surrounded by a pellicle. They They possess a large nucleus, nucleus, nuare oval to elongate and merous ribosomes, micro tubules, a mitochondrion with tubular cristae, a cytostome, an merous apical complex consisting of three polar rings, electron-dense paired rhoptries, and micronemes. Mature gametocytes are irregular in shape and are surrounded by a threelayered pellicle, and and contain a large nucleus, nucleus, mitochondria with tubular cristae, endoand lipid lipid inclusions. plasmic reticulum, numerous ribosomes, food food vacuoles, cytostomes, and plasmic Macrogametocytes are easily distinguished from from the microgametocytes by more more ribosome concentration and the presence of well developed endoplasmatic reticulum.
Introduction
Leger (1909) first discovered Leucocytozoon Leucocytozoon (Akiba) caulleryi Since Mathis and Leger in the blood of domestic fowls in Vietnam in Southeast Asia, the morphological observations on the developmental stages of L. caulleryi in each host have been performed by light microscope. (Aikawa et al., The fine structure of the merozoites or gametocytes of L. simondi (Aikawa 1970; Desser, 1970, 1973; Desser et al., 1970; Sterling and Aikawa 1973), L.smithi ziemanni (Kocan and Kocan, 1978) has been (Milhous and Solis, 1973), and L. ziemanni (Milhous described. However, no previous ultrastructural research has been reported on L. caulleryi. The present report was designed to investigate the fine structure of the merornero"'f Presented at the 3 rd German-Japanese Cooperative Symposium on Protozoan Diseases, Kyoto, Japan, Oct. 28.-29. 1980.
Fine Structure of Leucocytozoon Leucocytozoon caulleryi caulleryi Fine
199 199
zoites and gametocytes of 1. L. caulleryi and to compare their fine structure with zoites stage of other Leucocytozoon species and other related haemosporieach similar stage dian parasites. Materials and Methods
caulleryi which had The present study was carried out using using the Shizuoka strain of L. caulleryi cyclic transmission in White Leghorn chickens chickens and colonized Culicoides Culicoides been maintained by cyclictransmission 1968 a) as a vector of L. caulleryi. Male chickens served as arakawae (Morii and Kitaoka, 1968a) arakawae feeding of C. C. arakawae arakawae for infection with L. caulleryi caulleryi experimental birds. The procedures of feeding and the preparation of sporozoites for inoculation to chickens chickens were the same as described in the previous reports (Morii and Kitaoka, Kitaoka, 1968a, 1968 a, 1968b, 1968 b, 1968 c, 1969). 1969). 10 3 sporozoites. The Ten 30-day-old chickens were inoculated intravenously with 1 x 10 vein of inperipheral blood samples samples containing merozoites merozoites were collected collected from the wing vein chickens on the 14th and 15th day after sporozoite inoculation. The blood samples fected chickens chickens on the 19th and containing mature gametocytes were obtained from the infected chickens day. 20th day. merozoites or gametocytes was fixed °C in Each blood sample containing merozoites fixed for 2 hr at 4 °C in 2.5% glutaraldehyde and 2% formaldehyde in 0.2 M phosphate buffer at pH 7.4. After 2.5% 0.1 M phosphate buffer buffer containing 4% sucrose sucrose at pH 7.4, the samples samples were rinsing in 0.1 postfixed for 2 hr at 4 °C °C in % osmium in 11% osmium tetroxide tetroxide in 0.2 0.2 M phosphate phosphate buffer buffer at at pH pH 7.4, 7.4, ascending series series of alcohol and propylene oxide. The and subsequently dehydrated in an ascending samples 812. Sections Sections were cut with a Porter-Blum Porter-Blum MT-2 ultrasamples were embedded in Epon 812. microtome, stained with uranyl acetate and lead citrate, and examined in a Nihon Denshi 100B electron microscope. microscope. 100B
Results Schizonts Schizonts of L. caulleryi in infected chickens matured and released their merozoites into the bloodstream on the 14th day after sporozoite inoculation. Numerous exoerythrocytic or erythrocytic merozoites were found in the peripheral blood of chickens on the 14th or 15th day. Exoerythrocytic merozoites are oval to elongate and are surrounded by a pelthin outer and thick inner membrane (Fig. 1, 2). A licle which is composed of a thin round to oval nucleus is located in the central or posterior portion of the exoerythrocytic merozoite. Merozoites contain numerous ribosomes, a mitochondrion with tubular cristae, an apical complex consisting of three polar rings, electrondense (Fig. 1, 2, insert). Small dense paired rhoptries, microtubules, and micronemes (Fig. the paired rhoptries. Riboelectron-dense oval micronemes are associated with the somes are abundant in the cytoplasm and are mainly of the free type. Subpellicular microtubules originating from the most distal polar ring extend posteriorly and the inner membrane complex (Fig. (Fig. 2, insert). are located just beneath the the pelA cytostome consisting of two dense concentric segments is present in the licle of the merozoite about midway between its anterior and posterior ends (Fig. 2). (Fig. Exoerythrocytic merozoites of L.. caulleryi penetrate into polychromatic and surmature erythrocytes. Erythrocytic merozoites are irregular in shape and are surrounded by an additional membrane probably derived from the plasmalemma of host cell. They are located within a parasitophorous vacuole (Fig. (Fig. 3). The structure
200
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ifr."",,,' "if
Fig. nucleus. Fig. 1. Exoerythrocytic merozoites containing a distinct nucleus. Abbreviations CT Cytostome MM Middle membrane MN Microneme ER Endoplasmatic Endoplasmaric reticulum FV Food vacuole MT Microtubule FV cell N Nucleus HC Cytoplasm of host cell OM Outer membrane Nucleus of host cell cell HN Nucleus PR Polar ring 1M 1M Inner membrane PV PV Parasitophorous vacuole L Lipid inclusion Mitochondrion R Rhoptry Mitochondrion M
of the erythrocytic merozoites is similar to that of the the exoerythrocytic merozoites. A prominent nucleus, a single mitochondrion with tubular cristae, and numerous the cytoplasm of the parasite. An apical ribosomes are seen in the apical complex consisting ring is of three polar rings and several microtubules originating from the polar ring observed in the anterior portion of the merozoite. A cytostome is also observed (Fig. 3). in the the posterior portion (Fig. L. caulleryi were found in the the Both mature macro- and microgametocytes of 1. peripheral blood of infected chickens between the 19th and 23rd day after sporozoite inoculation. zoite
Fig. 2. Exoerythrocytic merozoite showing a cytostome in the pellicle. pellicle. Fig. cons:sting of polar rings, microtubules, rhoptries, and microInsert: An apical complex consisting nemes in an excerythrocytic merozoite. nemes Fig. 3. Erythrocytic merozoite located within a parasitophorous vacuole. vacuole. Fig.
Fine Structure of Leucocytozoon caulleryi
1 ...
20: 20:
202
T.Morii, T.Matsui, T T.lijima, .lijima, and M.Fukuda
Fig. Fig. 4. Mature macrogametocyte containing numerous ribosomes and well developed endoplasmatic reticulum.
® three-layered Fig. 5. Mature macrogametocyte surrounded by a distinct three-la yered pellicle.
Mature gametocytes are enclosed by the cytoplasm of host cell, but the nucleus host cell is not observed around the mature gametocytes (Fig. 4, 6). of host Mature macrogametocytes are irregular in shape and are surrounded by a distinct three-layered pellicle which is composed of the outer, middle, and inner
Fine Structure of Leucocyrozoon Leucocytozoon caulleryi caulleryi Fine
203
®® Fig. cytostome in the pellicle. Fig. 6. Mature microgametocyte showing a cytostome
(Fig. 5), and contain randomly located membrane (Fig. contai n a compact nucleus, numerous nu merous ra ndo mly locat ed reticmitochondria with promiment prom iment tubular cristae, well developed develop ed endoplasmatic endo plasma tic ret iculum, num numerous ribosomes, cytostomes, foodd vacuoles (Fig. erou s riboso mes, lipid inclusions, cytosto mes, and foo thee 4, 5). The structure of the mature mature microgametocytes is different from that of th Thee mitochondria microgametocytes are found in agmacrogametocytes. Th mitocho ndria of the microgarnetocytes nucleus.. The microgametocytes is diffuse and gregates close to the nucleus T he nucleus of the microgarnetocytes observed larger than that of th thee macrogametocytes. A cytostome is obse rved in the pellicle of the microgametocyte (Fig. 6). Macrogametocytes their M acrogametocytes are easily distinguished from the microgametocytes by the ir concentration dark appearance because of the more ribosome con centrat ion and the presence endoplasmatic reticulum. of a well developed endoplasrnatic Discussion
resemblee Thee fine struc structure gametocytes Th ture of the merozoites and gametocy tes of L. caulleryi resembl al., 1970; Desser, 1970, 1973; Desser et aI., al., 1970; those of L. simondi (Aikall'a (Aikawa et aI., L. smithi (Milhous (Milhous and Solis, So/is, 1973), and 1. L. ziemanni Sterling and Aikawa, 1973), 1. pre sence of a cytostome in the merozoites mero zoites (Kocan and Kocan, 1978), except for the presence
204
T.Morii, T.Matsui, T.Iijima, and M.Fukuda
L. and the disappearance of host cell nucleus close to mature gametocytes of 1.
caulleryi.
not been observed in the merozoites of 1. L. simondi, but it is A cytostome had not L. caulleryi. However the cytostomes of the gamepresent in the merozoites of 1. L. simondi (Desser et aI., a!., 1970; Sterling and Aikawa, 1973) 1973) and 1. L. caultocytes of 1. leryi have been observed. The structure of the cytostome of the merozoites of leryi L. caulleryi resembles that of the merozoites of Plasmodium (Aikawa (Aikawa et aI., 1966, 1. 1967) and Haemoproteus Haemoproteus (Sterling and DeGiusti, DeGiusti, 1972). The structure of the cyto1967) L. caulleryi is similar to that of 1. L. simondi. simondi. stome of the gametocyte of 1. Food vacuoles are readily identifiable within the cytoplasm of the erythrocytic stages of Plasmodium (Aikawa (Aikawa et aI., a!., 1969; Sterling and Aikawa, 1973) and HaemoHaemo1973; proteus proteus (Bradbury and Trager, 1968; Sterling, 1972; Sterling and Aikawa, 1973; Gallucci,1974). Gallucci, 1974). Food vacuoles are difficult to recognize in the erythrocytic merozoites and gametocytes of 1. L. caulleryi due to the absence of electron-dense food vacuoles and residual pigment which have been observed in Plasmodium Plasmodium and Haemoproteus Haemoproteus a!., 1966, 1969; Bradbury Bradbury and Trager, 1968; Sterling and Aikawa, (Aikawa et aI., (Aikawa 1973). This result suggests that the metabolic requirements of the erythrocytic stages of 1. L. caulleryi are different from those of Plasmodium Plasmodium and Haemoproteus. (1970) and Desser et al. (1970) (1970) reported that male and female merozoites Desser (1970) L. simondi simolldi can be distinguished by the difference in the density of ribosomes of 1. in their cytoplasm. However, there is no difference in the density of ribosomes in L. caulleryi, and it is difficult to distinguish the cytoplasm of each merozoite of 1. between male and female merozoites. L. caulleryi are enclosed by an additional membrane Erythrocytic merozoites of 1. probably derived from the plasmalemma of host cell. cell. This result indicates that L. caulleryi may invade the host cell by the same method shown the merozoites of 1. Plasmodium (Ladda et aI., a!., 1969). in Plasmodium L. caulleryi are enclosed by the cytoplasm of host cell, Mature gametocytes of 1. not observed close to the gametocytes. This structural but the nucleus of host cell is not L. caulleryi is different from that of other feature of the mature gametocytes of 1. species. Leucocytozoon species. L. caulleryi is more basophilic The cytoplasm of the macrogametocytes of 1. than the microgametocytes when stained with Giemsa's stain. This may be related to the difference in the density of cytoplasmic ribosomes. A darker cytoplasm containing numerous ribosomes is one of the features which distinguishes the L. caulleryi. macrogametocytes from the microgametocytes of 1. References 1. Aikawa, M., P. K. Hepler, C. G. Huff, and H. Sprinz: The feeding mechnism of avian 1. J. Cell Bio!' Bio!. 28 (1966) (1966) 355-373 malarial parasites. J. Plas2. Aikawa, M., C.G.Huff, and H.Sprinz: Fine structure of the asexual stages of PlasJ. Cell Bio!' Bio!. 34 (1967) (1967) 229-249 modium elongatum. J. H.Sprinz: 3. Aikawa, M., C.G.Huff, and Hi Sprinz: Comparative fine fine structure study of the gameparasites. J. J. Ultrastruct. Res. 26 tocytes of avian, reptilian, and mammalian malarial parasites. (1969) 316-331 (1969) C. P. A. Strome: Morphological study of microgarnerogenemicrogametogene4. Aikawa, M., C. G. Huff, and C.P. (1970) 43-68 43-68 sis of Leucocytozoon simondi. ]. Ultrastruct. Res. 32 (1970)
caulleryi Fine Structure of Leucocytozoon ca ulleryi
205
5. Brad Bradbury, Trager: structure thee mature ga gametes of H Haemobury, P. C. and W. Tra ger: The fine st ructu re of th metes of aem oproteus columbae Kruse. Protozool. 89-102 prot eus colum bae Kru se. JJ.. Prot ozool, 15 (1968) 89102 Thee fine structure structure of Leucocyt Leucocytozooll 6. Desser, S. S.: Th ozoon simondi. II. Megaloschizogony. Megaloschizogon y. Zool. 48 (1970) (1970) 417-421 Canad. J. Zoo!' 77.. Desser, S. S., ]. ] . R. r, and P. Lake: The fine str uct ure of Leucocyto zoo n simondi. R. Bake Baker, structure Leucocytozoon simolldi. I. Ga Gametocytogenesis. J. Zo Zool. (1970) 331-336 metocytogenes is. Can ad. J. o!' 48 (1970) S.:: The fine structure of Leucocytozooll simolldi. VI. Hep Hepatic schizogony. 8. Desser, S. S. Leucocyt ozoon simondi. ati c schizogo ny. J. Zoo!' Zool. 51 51 (1973) (1973) 605-609 Canad. Ca na d. J. structure Haemoproteus 9. Gallucci, B. B. B.: Fine stru ctu re of Haem oproteus columbae Kruse during macrogametomacrogam etofertilization. genesis and fertilizati on . J. J . Protozool. Prot ozoo!. 21 21 (1974) 254-263 A.A. K.M.Kocan: structure elongate gametocytes 10. Kocan, A. A. and K. M . Kocan : The fine struc ture of elonga te ga metocy tes of LeucoJ. Para Parasit. cytozooll cyt ozo on ziemanni (Laveran). J. sir. 64 (1978) 1057-1059 1057-1 059 R.L., M M.Aikawa, H.SpriIlZ: 11. Ladda, R.I., .Aikawa, and Hi Sprinz : Penetration of erythrocytes by merozoites of mammalian and avian malarial para parasites. J. Parasit. 55 (1969) (1969) 633-644 sites. J. (1909) 470-472 12. Mathis, C. and and M. Leger: Leucocytozoon Leucocyt ozoon du poulet. C. R. Soc. BioI. Bio!. 67 (1909) infection Leltco13. Milhous, W. and ]. ]. Solis: Turkey Leucocytozoon infect ion 3. Ultrastructure Ultr astructure of Leucocytozooll smithi gametocytes. Poult. Sci. 52 (1973) (1973) 2138-2146 cytozoon Kitaoka la boratory arakaU'ae (Diptera:: 14. Morii, T. and S. Kit aok a : The lab or atory colonization of Culicoides arak au iae (Diptera 26-40 Ceratopogonidae). Inst, Anim. Hlth Quart. 8 (1968a) 2640 Cera topogonidae). Nat. Inst. Morii, gametocyte 15. Mo rii, T. and S. Kitaoka: Kitaok a : Relationship Relat ion ship between the course of ga metocyte appearance caltlleryi in th thee chicken and spor sporozoite production arakml'ae. of Akiba caulleryi ozoite product ion in Culicoides arak awae. Nat.r. Inst. Anim.. Hlth Quart. (1968 b) 204204-209 Na Inst, Anim Q uart. 8 (1968b) 209 Kitaokd:: Influence of temperature on the spo sporogony cdulleryi 16. Morii, T. and S. Kitaoka rogo ny of Akiba caulleryi species.. Nat. in three Cltlicoides Culicoides species Nat . Inst. Insr. Anim. Hlth Quart. Qu art . 88 (1968c) (l968c) 210-216 Kitaoka: Susceptibility to sporo sporozoites 17. Morii, T. and S. Kitao ka : Suscepti bility of chickens of different ages to zoites of Akiba caul/eryii at various doses. Na Nat. Inst., Anim. Ak iba caullery r. Inst Anim . Hlth Quart. Q uart. 9 (1969) 104-111 18. Sterling, C. R R.: study gametogenesis HaenlO . : Ultrastructure stu dy of gametocytes and ga metoge nesis of H aem o-Protozool. 69-76 metclmikol'i. in, J. Prot ozoo!. 19 (1972) 6976 proteus metcbniko 19. Ste Sterling, and L. De Giust Giusti: Ultrastructural rling, C. R. an d D. I. i: Ultr astru ctura l aspects of schizogony, mature schiand merozoites metchnikol'i. J. J. Pa Paras it. 58 (1972) (1972) 641-652 zonts, an d mero zoites of Haemoproteus metchnihoui, rasit. M.. Aikawa comparative study gametocyte 20. Sterling, C. R. and M Aikawa:: A compa rative st udy of ga metoc yte ultrastructure in avian haemosporidia. J. J. Protozool. Prot ozoo!. 20 (1973) 81-92
Dr. T. Morii, Parasi tology, T.Mo rii, Dept. Depr. of Parasitol ogy, School of Medicine, M edicine, Kyorin University, Universi ty, Mitaka, Mir ak a, Tokyo Japann T okyo 181, Japa
Department of Parasitology, School of Medicine, Kyorin University, Mitaka, Tokyo 181, Japan
The Fine Structure of the Merozoites and Gametocytes of Leucocytozoon caulleryi * UJIMA, and TSUTOMU MORU, TOSHIHIRO MATSUI, TOSHIHIKO UlIMA, MINORU FUKUDA
Figures· Received February 10, 1981 With 6 Figures' 1981
Summary The fine structure of the merozoites and and gametocytes of Leucocytozoon caulleryi from from The the peripheral blood of chickens was studied by means of electron microscopy. Merozoites and are surrounded by a pellicle. They They possess a large nucleus, nucleus, nuare oval to elongate and merous ribosomes, micro tubules, a mitochondrion with tubular cristae, a cytostome, an merous apical complex consisting of three polar rings, electron-dense paired rhoptries, and micronemes. Mature gametocytes are irregular in shape and are surrounded by a threelayered pellicle, and and contain a large nucleus, nucleus, mitochondria with tubular cristae, endoand lipid lipid inclusions. plasmic reticulum, numerous ribosomes, food food vacuoles, cytostomes, and plasmic Macrogametocytes are easily distinguished from from the microgametocytes by more more ribosome concentration and the presence of well developed endoplasmatic reticulum.
Introduction
Leger (1909) first discovered Leucocytozoon Leucocytozoon (Akiba) caulleryi Since Mathis and Leger in the blood of domestic fowls in Vietnam in Southeast Asia, the morphological observations on the developmental stages of L. caulleryi in each host have been performed by light microscope. (Aikawa et al., The fine structure of the merozoites or gametocytes of L. simondi (Aikawa 1970; Desser, 1970, 1973; Desser et al., 1970; Sterling and Aikawa 1973), L.smithi ziemanni (Kocan and Kocan, 1978) has been (Milhous and Solis, 1973), and L. ziemanni (Milhous described. However, no previous ultrastructural research has been reported on L. caulleryi. The present report was designed to investigate the fine structure of the merornero"'f Presented at the 3 rd German-Japanese Cooperative Symposium on Protozoan Diseases, Kyoto, Japan, Oct. 28.-29. 1980.
Fine Structure of Leucocytozoon Leucocytozoon caulleryi caulleryi Fine
199 199
zoites and gametocytes of 1. L. caulleryi and to compare their fine structure with zoites stage of other Leucocytozoon species and other related haemosporieach similar stage dian parasites. Materials and Methods
caulleryi which had The present study was carried out using using the Shizuoka strain of L. caulleryi cyclic transmission in White Leghorn chickens chickens and colonized Culicoides Culicoides been maintained by cyclictransmission 1968 a) as a vector of L. caulleryi. Male chickens served as arakawae (Morii and Kitaoka, 1968a) arakawae feeding of C. C. arakawae arakawae for infection with L. caulleryi caulleryi experimental birds. The procedures of feeding and the preparation of sporozoites for inoculation to chickens chickens were the same as described in the previous reports (Morii and Kitaoka, Kitaoka, 1968a, 1968 a, 1968b, 1968 b, 1968 c, 1969). 1969). 10 3 sporozoites. The Ten 30-day-old chickens were inoculated intravenously with 1 x 10 vein of inperipheral blood samples samples containing merozoites merozoites were collected collected from the wing vein chickens on the 14th and 15th day after sporozoite inoculation. The blood samples fected chickens chickens on the 19th and containing mature gametocytes were obtained from the infected chickens day. 20th day. merozoites or gametocytes was fixed °C in Each blood sample containing merozoites fixed for 2 hr at 4 °C in 2.5% glutaraldehyde and 2% formaldehyde in 0.2 M phosphate buffer at pH 7.4. After 2.5% 0.1 M phosphate buffer buffer containing 4% sucrose sucrose at pH 7.4, the samples samples were rinsing in 0.1 postfixed for 2 hr at 4 °C °C in % osmium in 11% osmium tetroxide tetroxide in 0.2 0.2 M phosphate phosphate buffer buffer at at pH pH 7.4, 7.4, ascending series series of alcohol and propylene oxide. The and subsequently dehydrated in an ascending samples 812. Sections Sections were cut with a Porter-Blum Porter-Blum MT-2 ultrasamples were embedded in Epon 812. microtome, stained with uranyl acetate and lead citrate, and examined in a Nihon Denshi 100B electron microscope. microscope. 100B
Results Schizonts Schizonts of L. caulleryi in infected chickens matured and released their merozoites into the bloodstream on the 14th day after sporozoite inoculation. Numerous exoerythrocytic or erythrocytic merozoites were found in the peripheral blood of chickens on the 14th or 15th day. Exoerythrocytic merozoites are oval to elongate and are surrounded by a pelthin outer and thick inner membrane (Fig. 1, 2). A licle which is composed of a thin round to oval nucleus is located in the central or posterior portion of the exoerythrocytic merozoite. Merozoites contain numerous ribosomes, a mitochondrion with tubular cristae, an apical complex consisting of three polar rings, electrondense (Fig. 1, 2, insert). Small dense paired rhoptries, microtubules, and micronemes (Fig. the paired rhoptries. Riboelectron-dense oval micronemes are associated with the somes are abundant in the cytoplasm and are mainly of the free type. Subpellicular microtubules originating from the most distal polar ring extend posteriorly and the inner membrane complex (Fig. (Fig. 2, insert). are located just beneath the the pelA cytostome consisting of two dense concentric segments is present in the licle of the merozoite about midway between its anterior and posterior ends (Fig. 2). (Fig. Exoerythrocytic merozoites of L.. caulleryi penetrate into polychromatic and surmature erythrocytes. Erythrocytic merozoites are irregular in shape and are surrounded by an additional membrane probably derived from the plasmalemma of host cell. They are located within a parasitophorous vacuole (Fig. (Fig. 3). The structure
200
T. Morii, T. Matsui, T.lijima, T.Iijima, and M. Fukuda ~.".".,,'
ifr."",,,' "if
Fig. nucleus. Fig. 1. Exoerythrocytic merozoites containing a distinct nucleus. Abbreviations CT Cytostome MM Middle membrane MN Microneme ER Endoplasmatic Endoplasmaric reticulum FV Food vacuole MT Microtubule FV cell N Nucleus HC Cytoplasm of host cell OM Outer membrane Nucleus of host cell cell HN Nucleus PR Polar ring 1M 1M Inner membrane PV PV Parasitophorous vacuole L Lipid inclusion Mitochondrion R Rhoptry Mitochondrion M
of the erythrocytic merozoites is similar to that of the the exoerythrocytic merozoites. A prominent nucleus, a single mitochondrion with tubular cristae, and numerous the cytoplasm of the parasite. An apical ribosomes are seen in the apical complex consisting ring is of three polar rings and several microtubules originating from the polar ring observed in the anterior portion of the merozoite. A cytostome is also observed (Fig. 3). in the the posterior portion (Fig. L. caulleryi were found in the the Both mature macro- and microgametocytes of 1. peripheral blood of infected chickens between the 19th and 23rd day after sporozoite inoculation. zoite
Fig. 2. Exoerythrocytic merozoite showing a cytostome in the pellicle. pellicle. Fig. cons:sting of polar rings, microtubules, rhoptries, and microInsert: An apical complex consisting nemes in an excerythrocytic merozoite. nemes Fig. 3. Erythrocytic merozoite located within a parasitophorous vacuole. vacuole. Fig.
Fine Structure of Leucocytozoon caulleryi
1 ...
20: 20:
202
T.Morii, T.Matsui, T T.lijima, .lijima, and M.Fukuda
Fig. Fig. 4. Mature macrogametocyte containing numerous ribosomes and well developed endoplasmatic reticulum.
® three-layered Fig. 5. Mature macrogametocyte surrounded by a distinct three-la yered pellicle.
Mature gametocytes are enclosed by the cytoplasm of host cell, but the nucleus host cell is not observed around the mature gametocytes (Fig. 4, 6). of host Mature macrogametocytes are irregular in shape and are surrounded by a distinct three-layered pellicle which is composed of the outer, middle, and inner
Fine Structure of Leucocyrozoon Leucocytozoon caulleryi caulleryi Fine
203
®® Fig. cytostome in the pellicle. Fig. 6. Mature microgametocyte showing a cytostome
(Fig. 5), and contain randomly located membrane (Fig. contai n a compact nucleus, numerous nu merous ra ndo mly locat ed reticmitochondria with promiment prom iment tubular cristae, well developed develop ed endoplasmatic endo plasma tic ret iculum, num numerous ribosomes, cytostomes, foodd vacuoles (Fig. erou s riboso mes, lipid inclusions, cytosto mes, and foo thee 4, 5). The structure of the mature mature microgametocytes is different from that of th Thee mitochondria microgametocytes are found in agmacrogametocytes. Th mitocho ndria of the microgarnetocytes nucleus.. The microgametocytes is diffuse and gregates close to the nucleus T he nucleus of the microgarnetocytes observed larger than that of th thee macrogametocytes. A cytostome is obse rved in the pellicle of the microgametocyte (Fig. 6). Macrogametocytes their M acrogametocytes are easily distinguished from the microgametocytes by the ir concentration dark appearance because of the more ribosome con centrat ion and the presence endoplasmatic reticulum. of a well developed endoplasrnatic Discussion
resemblee Thee fine struc structure gametocytes Th ture of the merozoites and gametocy tes of L. caulleryi resembl al., 1970; Desser, 1970, 1973; Desser et aI., al., 1970; those of L. simondi (Aikall'a (Aikawa et aI., L. smithi (Milhous (Milhous and Solis, So/is, 1973), and 1. L. ziemanni Sterling and Aikawa, 1973), 1. pre sence of a cytostome in the merozoites mero zoites (Kocan and Kocan, 1978), except for the presence
204
T.Morii, T.Matsui, T.Iijima, and M.Fukuda
L. and the disappearance of host cell nucleus close to mature gametocytes of 1.
caulleryi.
not been observed in the merozoites of 1. L. simondi, but it is A cytostome had not L. caulleryi. However the cytostomes of the gamepresent in the merozoites of 1. L. simondi (Desser et aI., a!., 1970; Sterling and Aikawa, 1973) 1973) and 1. L. caultocytes of 1. leryi have been observed. The structure of the cytostome of the merozoites of leryi L. caulleryi resembles that of the merozoites of Plasmodium (Aikawa (Aikawa et aI., 1966, 1. 1967) and Haemoproteus Haemoproteus (Sterling and DeGiusti, DeGiusti, 1972). The structure of the cyto1967) L. caulleryi is similar to that of 1. L. simondi. simondi. stome of the gametocyte of 1. Food vacuoles are readily identifiable within the cytoplasm of the erythrocytic stages of Plasmodium (Aikawa (Aikawa et aI., a!., 1969; Sterling and Aikawa, 1973) and HaemoHaemo1973; proteus proteus (Bradbury and Trager, 1968; Sterling, 1972; Sterling and Aikawa, 1973; Gallucci,1974). Gallucci, 1974). Food vacuoles are difficult to recognize in the erythrocytic merozoites and gametocytes of 1. L. caulleryi due to the absence of electron-dense food vacuoles and residual pigment which have been observed in Plasmodium Plasmodium and Haemoproteus Haemoproteus a!., 1966, 1969; Bradbury Bradbury and Trager, 1968; Sterling and Aikawa, (Aikawa et aI., (Aikawa 1973). This result suggests that the metabolic requirements of the erythrocytic stages of 1. L. caulleryi are different from those of Plasmodium Plasmodium and Haemoproteus. (1970) and Desser et al. (1970) (1970) reported that male and female merozoites Desser (1970) L. simondi simolldi can be distinguished by the difference in the density of ribosomes of 1. in their cytoplasm. However, there is no difference in the density of ribosomes in L. caulleryi, and it is difficult to distinguish the cytoplasm of each merozoite of 1. between male and female merozoites. L. caulleryi are enclosed by an additional membrane Erythrocytic merozoites of 1. probably derived from the plasmalemma of host cell. cell. This result indicates that L. caulleryi may invade the host cell by the same method shown the merozoites of 1. Plasmodium (Ladda et aI., a!., 1969). in Plasmodium L. caulleryi are enclosed by the cytoplasm of host cell, Mature gametocytes of 1. not observed close to the gametocytes. This structural but the nucleus of host cell is not L. caulleryi is different from that of other feature of the mature gametocytes of 1. species. Leucocytozoon species. L. caulleryi is more basophilic The cytoplasm of the macrogametocytes of 1. than the microgametocytes when stained with Giemsa's stain. This may be related to the difference in the density of cytoplasmic ribosomes. A darker cytoplasm containing numerous ribosomes is one of the features which distinguishes the L. caulleryi. macrogametocytes from the microgametocytes of 1. References 1. Aikawa, M., P. K. Hepler, C. G. Huff, and H. Sprinz: The feeding mechnism of avian 1. J. Cell Bio!' Bio!. 28 (1966) (1966) 355-373 malarial parasites. J. Plas2. Aikawa, M., C.G.Huff, and H.Sprinz: Fine structure of the asexual stages of PlasJ. Cell Bio!' Bio!. 34 (1967) (1967) 229-249 modium elongatum. J. H.Sprinz: 3. Aikawa, M., C.G.Huff, and Hi Sprinz: Comparative fine fine structure study of the gameparasites. J. J. Ultrastruct. Res. 26 tocytes of avian, reptilian, and mammalian malarial parasites. (1969) 316-331 (1969) C. P. A. Strome: Morphological study of microgarnerogenemicrogametogene4. Aikawa, M., C. G. Huff, and C.P. (1970) 43-68 43-68 sis of Leucocytozoon simondi. ]. Ultrastruct. Res. 32 (1970)
caulleryi Fine Structure of Leucocytozoon ca ulleryi
205
5. Brad Bradbury, Trager: structure thee mature ga gametes of H Haemobury, P. C. and W. Tra ger: The fine st ructu re of th metes of aem oproteus columbae Kruse. Protozool. 89-102 prot eus colum bae Kru se. JJ.. Prot ozool, 15 (1968) 89102 Thee fine structure structure of Leucocyt Leucocytozooll 6. Desser, S. S.: Th ozoon simondi. II. Megaloschizogony. Megaloschizogon y. Zool. 48 (1970) (1970) 417-421 Canad. J. Zoo!' 77.. Desser, S. S., ]. ] . R. r, and P. Lake: The fine str uct ure of Leucocyto zoo n simondi. R. Bake Baker, structure Leucocytozoon simolldi. I. Ga Gametocytogenesis. J. Zo Zool. (1970) 331-336 metocytogenes is. Can ad. J. o!' 48 (1970) S.:: The fine structure of Leucocytozooll simolldi. VI. Hep Hepatic schizogony. 8. Desser, S. S. Leucocyt ozoon simondi. ati c schizogo ny. J. Zoo!' Zool. 51 51 (1973) (1973) 605-609 Canad. Ca na d. J. structure Haemoproteus 9. Gallucci, B. B. B.: Fine stru ctu re of Haem oproteus columbae Kruse during macrogametomacrogam etofertilization. genesis and fertilizati on . J. J . Protozool. Prot ozoo!. 21 21 (1974) 254-263 A.A. K.M.Kocan: structure elongate gametocytes 10. Kocan, A. A. and K. M . Kocan : The fine struc ture of elonga te ga metocy tes of LeucoJ. Para Parasit. cytozooll cyt ozo on ziemanni (Laveran). J. sir. 64 (1978) 1057-1059 1057-1 059 R.L., M M.Aikawa, H.SpriIlZ: 11. Ladda, R.I., .Aikawa, and Hi Sprinz : Penetration of erythrocytes by merozoites of mammalian and avian malarial para parasites. J. Parasit. 55 (1969) (1969) 633-644 sites. J. (1909) 470-472 12. Mathis, C. and and M. Leger: Leucocytozoon Leucocyt ozoon du poulet. C. R. Soc. BioI. Bio!. 67 (1909) infection Leltco13. Milhous, W. and ]. ]. Solis: Turkey Leucocytozoon infect ion 3. Ultrastructure Ultr astructure of Leucocytozooll smithi gametocytes. Poult. Sci. 52 (1973) (1973) 2138-2146 cytozoon Kitaoka la boratory arakaU'ae (Diptera:: 14. Morii, T. and S. Kit aok a : The lab or atory colonization of Culicoides arak au iae (Diptera 26-40 Ceratopogonidae). Inst, Anim. Hlth Quart. 8 (1968a) 2640 Cera topogonidae). Nat. Inst. Morii, gametocyte 15. Mo rii, T. and S. Kitaoka: Kitaok a : Relationship Relat ion ship between the course of ga metocyte appearance caltlleryi in th thee chicken and spor sporozoite production arakml'ae. of Akiba caulleryi ozoite product ion in Culicoides arak awae. Nat.r. Inst. Anim.. Hlth Quart. (1968 b) 204204-209 Na Inst, Anim Q uart. 8 (1968b) 209 Kitaokd:: Influence of temperature on the spo sporogony cdulleryi 16. Morii, T. and S. Kitaoka rogo ny of Akiba caulleryi species.. Nat. in three Cltlicoides Culicoides species Nat . Inst. Insr. Anim. Hlth Quart. Qu art . 88 (1968c) (l968c) 210-216 Kitaoka: Susceptibility to sporo sporozoites 17. Morii, T. and S. Kitao ka : Suscepti bility of chickens of different ages to zoites of Akiba caul/eryii at various doses. Na Nat. Inst., Anim. Ak iba caullery r. Inst Anim . Hlth Quart. Q uart. 9 (1969) 104-111 18. Sterling, C. R R.: study gametogenesis HaenlO . : Ultrastructure stu dy of gametocytes and ga metoge nesis of H aem o-Protozool. 69-76 metclmikol'i. in, J. Prot ozoo!. 19 (1972) 6976 proteus metcbniko 19. Ste Sterling, and L. De Giust Giusti: Ultrastructural rling, C. R. an d D. I. i: Ultr astru ctura l aspects of schizogony, mature schiand merozoites metchnikol'i. J. J. Pa Paras it. 58 (1972) (1972) 641-652 zonts, an d mero zoites of Haemoproteus metchnihoui, rasit. M.. Aikawa comparative study gametocyte 20. Sterling, C. R. and M Aikawa:: A compa rative st udy of ga metoc yte ultrastructure in avian haemosporidia. J. J. Protozool. Prot ozoo!. 20 (1973) 81-92
Dr. T. Morii, Parasi tology, T.Mo rii, Dept. Depr. of Parasitol ogy, School of Medicine, M edicine, Kyorin University, Universi ty, Mitaka, Mir ak a, Tokyo Japann T okyo 181, Japa