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Protist, Vol. 157, 13—19, January 2006 http://www.elsevier.de/protis Published online date 20 January 2006
Ultrastructural Study of the Gametocytes and Merogonic Stages of Fallisia audaciosa (Haemosporina: Garniidae) that Infect Neutrophils of the Lizard Plica umbra (Reptilia: Iguanidae) Edilene O. Silvaa,1, Jose´ Antonio P. Dinizb, Ralph Lainsonc, Renato A. DaMattad, and Wanderley de Souzae a
Laborato´rio de Parasitologia, Departamento de Patologia, Centro de Cieˆncias Biolo´gicas, Universidade Federal do Para´, Av. Augusto Correˆa s/n, Bairro Guama´, 66075-110 Bele´m, Para´, Brazil b Unidade de Microscopia Eletroˆnica, Instituto Evandro Chagas, Av. Almirante Barroso 492, Bairro Marco, 66090-000 Bele´m, Para´, Brazil c Laborato´rio de Coccı´dios, Departamento de Parasitologia, Instituto Evandro Chagas, Av. Almirante Barroso 492, Bairro Marco, 66090-000 Bele´m, Para´, Brazil d Laborato´rio de Biologia Celular e Tecidual, Centro de Biocieˆncias e Biotecnologia, Universidade Estadual do Norte Fluminense, Avenida Alberto Lamego 2000, 28013-600 Campos dos Goytacazes, RJ, Brazil e Laborato´rio de Ultraestrutura Celular Hertha Meyer, Instituto de Biofı´sica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Ilha do Funda˜o, 21941-590 Rio de Janeiro, RJ, Brazil Submitted June 10, 2005; Accepted October 30, 2005 Monitoring Editor: C. Graham Clark
Little is known regarding the ultrastructure of the genus Fallisia (Apicomplexa: Haemosporina: Garniidae). This report describes the fine structure of some developmental stages of Fallisia audaciosa that infect neutrophils in the peripheral blood of the Amazonian lizard Plica umbra (Reptilia: Iguanidae). The parasites lie within a parasitophorous vacuole and exhibit the basic structures of members of the Apicomplexa, such as the pellicle and the cytostome. Invaginations of the inner membrane complex were seen in the gametocytes and may be concerned with nutrition. The meronts were irregularly shaped before division, a feature unusual among members of the Apicomplexa. The unusual presence of a parasitic protozoan within neutrophils, in some way interfering with or modulating the microbicidal activity of such cells, is discussed. & 2005 Elsevier GmbH. All rights reserved. Key words: Fallisia audaciosa; Garniidae; lizard; neutrophil; Plica umbra; ultrastructure.
Introduction Species of the genus Fallisia are apicomplexan parasites of lizards (Lainson et al. 1971, 1975) and, 1
Corresponding author; fax: +55 913201 7601. e-mail [email protected]
& 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.protis.2005.10.003
more rarely, birds (Gabaldon et al. 1985). All their developmental stages occur in leucocytes or thrombocytes of their vertebrate hosts (Lainson et al. 1971). Fallisia audaciosa infects the neutrophils of the iguanidae lizard Plica umbra (Lainson et al. 1975). Neutrophils, immune cells involved in the first line of defense, are capable of migrating to inflamed tissue to destroy bacteria, fungi, and
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protozoa by an impressive array of microbicidal mechanisms, such as production of reactive oxygen species and the release of cytotoxic molecules stored in their cytoplasmic granules (Faurschou and Borregaard 2003; Kobayashi et al. 2003). Although some reports describe that neutrophils are infected by a few microbial pathogens (Chen et al. 1994; Laskay et al. 2003; Laufs et al. 2002; Yoshiie et al. 2000), the only description of a protozoan multiplying in neutrophils appears to be that of F. audaciosa (Lainson et al. 1975). Although the morphology of F. audaciosa in the lizard host has been well documented by light microscopy (Lainson et al. 1975), there have been no studies of its fine structure. In this study, we have confirmed that the host cell of F. audaciosa is the neutrophil and describe the ultrastructure of the macrogametocyte and merogonic stages of the protozoan.
Results Observations by light microscopy of blood films of a highly infected lizard showed that 25% of the blood neutrophils were infected. Gametocytes, meronts, and merozoites were confined to neutrophils (Fig. 1A—C). Multiple invasion of neutrophils was not seen in the present material, although Lainson et al. (1975) noted that this was ‘‘y a marked feature in heavy infections’’. The nucleus of a few infected neutrophils was altered and the cells were enlarged, probably due to the advanced stage of infection (Fig. 1C). TEM examination showed that uninfected neutrophils were irregular in shape, with delicate membrane projections. The centrally located multilobulated nucleus contained large clumps of heterochromatin in close contact with the nuclear membrane. The cytoplasm contained vacuoles and a few granules (data not shown). Infected neutrophils showed some irregular projections and had parasites within a parasitophorous vacuole (Figs 2A,B, 3A,C,D). Only macrogametocytes and meronts were observed by TEM. The macrogametocytes had a three-layered pellicle composed of an outer and an inner membrane complex formed by two closely apposed membrane units (Fig. 2B, inset). Invaginations of the inner membrane complex of gametocytes were observed (Fig. 2C—E). A large vacuole, the contents of which were similar to that found in the host neutrophil cytoplasm, was found inside the parasite (Fig. 2E). In some of the
Figure 1. Giemsa-stained blood film showing a neutrophil of Plica umbra infected by Fallisia audaciosa. A. Infected neutrophils showing gametocytes (arrow). Note the lobulated nuclei of the host cells. B. Early meront (arrow). C. A mature meront (arrow) and an uninfected neutrophil (arrowhead). Bar ¼ 10 mm.
macrogametocytes, an apicoplast was seen (data not shown). At the beginning of segmentation, meronts were irregular in shape (Fig. 3A,B). Mature forms had many nuclei (Fig. 3C). At the end of merogony, the merozoites within the parasitophorous vacuole were of heterogeneous density (Fig. 3D,E). In some of the developing merozoites, a structure resembling an apical complex was apparent, with
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Ultrastructure of Fallisia audaciosa 15
Figure 2. Transmission electron microscopy of peripheral blood neutrophils infected with Fallisia audaciosa. A. General view of an infected neutrophil (N) showing a macrogametocyte (MA). B. Section through a macrogametocyte (MA) infecting a neutrophil (N). Note the high electron-dense pellicle (arrows), see also inset (2 ). C. Invagination of the inner membrane complex (arrow). D. High magnification of a typical membrane system showing a cytostome (CY). E. Another image showing an invagination ending in a vacuole (arrow) containing material with similar electron density to that of the neutrophil cytoplasm. Neutrophil (N), parasite (P). Bars A, B ¼ 1 mm; C—E. Bar ¼ 0.5 mm.
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Figure 3. Transmission electron microscopy of the merogonic process of Fallisia audaciosa. A. The beginning of merogony (arrowheads). B. Higher magnification of A. C. Further stages of merogony. Note the numerous nuclei (asterisks). D. General view of an advanced stage of merogony with individual merozoites visible (arrows). E. Higher magnification of the merozoites (ME) showing structures suggestive of an apical complex (arrow). Note the different electron density of the merozoites. Neutrophil (N), parasite (P). Bar ¼ 1 mm.
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Ultrastructure of Fallisia audaciosa 17
organelles similar to rhoptries (Fig. 3E). However, polar ring and dense bodies were not observed.
Discussion The fine structure of some other Fallisia species has been described in some detail (Boulard et al. 1987; Silva et al. 2005). The results of this report provide new information regarding ultrastructural aspects of the species F. audaciosa infecting the neutrophils of the lizard P. umbra. TEM study of the parasite within the parasitophorous vacuole showed a complex pellicle system and apical structure suggesting the presence of an apical complex. Although these parasites can probably ingest nutrients from host cell cytoplasm through a cytostome, gametocytes also showed deep invaginations of the membrane complex that may be involved in nutrient intake by the parasite. A similar structure has been reported in gametocytes of another species of Fallisia, F. effusa (Silva et al. 2005), and also in Eimeria auburnensis (Hammond et al. 1967). Silva et al. (2004) also described a similar structure in a hematozoan, suspected to be a species of Lainsonia (Lainson et al. 2003) in the monocytes of the lizard Ameiva ameiva. In all cases, it was suggested that these invaginations might increase the cell surface for nutritional purposes. At the beginning of segmentation, the mature meront of F. audaciosa was seen to differ from other apicomplexans, in which there is usually segmentation of the thick inner membrane and the random appearance of rhoptries beneath and along the plasma membrane of the mother cell (Aikawa and Sterling 1974). Fallisia audaciosa is irregularly shaped before nuclear division, suggesting that the merogonic process is variable within the phylum Apicomplexa. In general, it is assumed that neutrophils are the first line of defense in vertebrates and are highly efficient in killing invading microorganisms (Faurschou and Borregaard 2003; Kobayashi et al. 2003). However, recent evidence has shown that some pathogens evade the microbicidal mechanisms or inhibit the spontaneous apoptosis of neutrophils (Aga et al. 2002; Laskay et al. 2003; Laufs et al. 2002; Yoshiie et al. 2000). Aga et al. (2002) showed that Leishmania promastigotes can modify spontaneous apoptosis of human neutrophils, but they do not replicate inside these cells. Gamonts of Hepatozoon canis were found to infect circulating neutrophils of dogs; however, no division stages were observed in these cells
(Droleskey et al. 1993). Only two pathogenic microorganisms have been shown to replicate within mammalian neutrophils (Herron et al. 2000; Van Zandbergen et al. 2004). One of them is Anaplasma phagocytophilum, which causes human granulocytic ehrlichiosis. This obligate intracellular bacterium is transmitted by ixodid tick bites, and can inhibit apoptosis, phagosome— lysosome fusion, and production of reactive oxygen species of neutrophils (Gokce et al. 1999; Mott and Rikihisa 2000; Scaife et al. 2003; Yoshiie et al. 2000). The other example is Chlamydia pneumoniae, which has been shown to delay apoptosis and to multiply inside neutrophils (Van Zandbergen et al. 2004). Apoptosis is an important mechanism in human neutrophils for the regulation of homeostasis and inflammation (Savill 1997). Fallisia audaciosa seems to be the only recorded protozoan that naturally infects and multiplies exclusively inside neutrophils, an achievement which prompted Lainson et al. (1975) to name it as F. audaciosa. The mechanisms responsible for this survival are not yet known; however, we can speculate that F. audaciosa probably delays the apoptotic process of the lizard neutrophil and inhibits the production of reactive oxygen species, enabling its survival in these cells. For a better understanding of this intriguing association, additional studies will be necessary to determine just how this parasite evades the microbicidal response of neutrophils.
Methods Lizards: During 2001 and 2003, 76 lizard specimens of Plica umbra were hand-captured from the trunks of trees in the tropical rain forest of Para´ State, north Brazil. They were maintained in an animal house of the Instituto Evandro Chagas and kept separately in standard mouse cages at 27 to 32 1C. They were provided with water and larvae and adults of Tenebrio molitor. Blood harvesting and leukocyte separation: Lizards were restrained manually and blood was collected by cardiac puncture into heparinized 1 ml syringes (100 U/ml). Blood smears from all lizards were fixed with absolute methanol and stained by Giemsa’s method. Blood leukocytes were separated using a method previously described (Silva et al. 2004). Light microscopy: The parasites were studied in Giemsa-stained blood smears, and photographed using a Zeiss Axiophote Microscope with the immersion 100 objective.
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Transmission electron microscopy: Blood buffy coat was fixed in 2.5% glutaraldehyde and 4% formaldehyde, in a buffer solution containing 60 mM Pipes, 20 mM Hepes, 10 mM ethyleneglycol-bis(B-aminoethylether)-N,N,N0 -tetraacetic acid, 70 mM KCl, and 5 mM MgCl2 (Schliwa and van Blerkom 1981), pH 6.9, for 1 h at room temperature. Afterward, cells were washed in the same buffer and post-fixed for 1 h in a solution containing 1% osmium tetroxide, 0.8% ferrocyanide, and 5 mM calcium chloride, washed, dehydrated in graded acetone, and embedded in epoxy resin. Thin sections were stained with uranyl acetate and lead citrate and examined in a Zeiss EM 900 transmission electron microscope.
Acknowledgements This work was supported by grants from Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Financiadora de Estudos e Projetos (FINEP), Programa de Nu´cleos de Exceleˆncia (PRONEX), Instituto Evandro Chagas/SVS/MS, Fundac- a˜o de Coordenac- a˜o de Pessoal de Nı´vel Superior (CAPES), Programa Nacional de Cooperac-a˜o Acadeˆmica (CAPES-PROCAD), Fundac- a˜o Carlos Chagas Filho de Amparo a` Pesquisa do Estado do Rio de Janeiro (FAPERJ), and the Wellcome Trust, London (RL). The authors thank Manoel CM de Souza and Antonio J de Oliveira Monteiro for their technical assistance and animal care, and Marcia A. Dutra and Eliandro Lima for their assistance with the photographic work. The experiments comply with current Brazilian animal protection laws (IBAMA doc. 02018.000301/02-13).
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