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Comparative ultrastructure of tachyzoites, bradyzoites, and tissue cysts of Neospora caninum and Toxoplasma gondii
International Journal for Parasitology 29 (1999) 1509±1519
Comparative ultrastructure of tachyzoites, bradyzoites, and tissue cysts of Neospora caninum and Toxoplasma gondii C.A. Speer a, *, J.P. Dubey b, M.M. McAllister c, J.A. Blixt a a
Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717-3610, USA United States Department of Agriculture, Agriculture Research Service, Livestock and Poultry Sciences Institute, Parasite Biology and Epidemiology Laboratory, BARC-East, Bldg. 1001, Beltsville, MD 20705-2350, USA c Department of Veterinary Pathology, University of Illinois, 2522 VMBSB, 2001 S. Lincoln Avenue, Urbana, IL 61802, USA
b
Received 29 April 1999; received in revised form 16 July 1999; accepted 16 July 1999
Abstract The ultrastructure of tachyzoites, bradyzoites and tissue cysts of the NC-1, NC-5 and NC-Liverpool strains of Neospora caninum are reviewed and compared with those of the VEG and ME-49 strains of Toxoplasma gondii. While each stage of N. caninum and T. gondii shared many ultrastructural characteristics, each parasite stage also had certain features or organelles that could be used to distinguish the two parasites. Some of the most prominent ultrastructural dierences occurred in the number, appearance and location of rhoptries, looped-back rhoptries, micronemes, dense granules, small dense granules and micropores. The tissue cysts of both parasites were also basically similar, being surrounded by a cyst wall and not compartmentalised by septa. The cyst wall of N. caninum was irregular and substantially thicker, 0.5±4 mm, than those of T. gondii which were smooth and 0.5 mm thick. # 1999 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Bradyzoites; Neospora caninum; Tachyzoites; Tissue cysts; Toxoplasma gondii; Ultrastructure
1. Introduction
2. Materials and methods
The tachyzoites, bradyzoites and tissue cysts of Neospora caninum and Toxoplasma gondii appear essentially identical by standard light microscopy. These parasites can, however, be dierentiated on the basis of certain ultrastructural features [1± 3]. In this paper, we review and compare the ultrastructure of the tachyzoites, bradyzoites and tissue cysts of N. caninum with those of T. gondii.
Tachyzoites of the NC-1 strain of N. caninum [4] were derived from cultured bovine monocytes (M617) [5] and bovine pulmonary artery endothelial cells (American Type Culture Collection). Tachyzoites of the VEG and ME-49 strains of T. gondii [6] were obtained from cultured M617 cells, CPA cells (ATCC), or the peritoneum of a Swiss Webster mouse that had been inoculated 6 days earlier with tachyzoites. Specimens were ®xed in 3% glutaraldehyde in Millonig's phosphate buer or modi®ed
* Corresponding author. Tel.: +1-406-994-6389; fax: +1406-994-6878.
0020-7519/99/$20.00 # 1999 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 0 - 7 5 1 9 ( 9 9 ) 0 0 1 3 2 - 0
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Figs. 1 and 2. Transmission EM of tachyzoites of N. caninum and T. gondii. Fig. 1. Tachyzoite of N. caninum NC-5 in a parasitophorous vacuole (PV) within a cultured M617 cell, showing various organelles and inclusion bodies. Some of the micronemes (Mn) are oriented perpendicularly to the zoite pellicle and the rhoptries are uniformly electron-dense, one of which (Rh) is oriented with its neck projecting posteriorly. Approximately 10 dense granules (Dg) are situated posterior and three anterior to the nucleus. Fig. 2. Tachyzoite of T. gondii VEG strain in a parasitophorous vacuole (PV) within a mouse peritoneal macrophage, showing labyrinthine rhoptries (Rh) and an early stage of endodyogeny in which a progeny zoite, consisting of a plastid (Ap*), Golgi complex (Go*), inner membrane complex (Im*), and rhoptries (Rh*), is developing internally within the mother zoite. Several host-cell mitochondria (*) are situated close to the parasitophorous vacuole. Abbreviations: Am, amylopectin; Ar 1, 2, apical rings 1 and 2; Co, conoid; Go, Golgi complex; Hc, host cell cytoplasm; Im, inner membrane complex of pellicle; Lb, lipid body; Mi, mitochondrion; Mn, microneme; No, nucleolus; Nu, nucleus; Pl, plasmalemma; Pr 1, 2, polar rings 1 and 2; Sm, subpellicular microtubule; Tm, tubulovesicular membrane network.
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Karnovsky's ®xative, rinsed in Millonig's phosphate buer or cacodylate buer, post-®xed in 1% osmium tetroxide, rinsed in buer, dehydrated in ethanol, and embedded in Spurr's medium. Some of the specimens were prestained with 1% uranyl acetate and 1% phosphotungstic acid in 70% ethanol overnight, prior to complete dehydration and embedding. Before examination in a JEOL 100CX TEM, ultrathin sections of prestained specimens were stained with lead citrate; non-prestained specimens were stained with uranyl acetate and lead citrate. Tissue cysts of N. caninum and T. gondii were obtained from infected brains and had been ®xed in Karnovsky's ®xative. Six tissue cysts were obtained from parts of the brain of a dog with NC-isolate [7]. Five N. caninum NC-Liverpool tissue cysts studied had been freed from the brains of infected mice using Percoll [8]. The mice had been inoculated with tachyzoites of the NC-Liverpool strain of N. caninum [9]. Tissue cysts were washed free of Percoll, and prepared for TEM as described above. Tissue cysts of the VEG or ME-49 strain of T. gondii were obtained from Swiss Webster mice that had been inoculated 7±9 months earlier with tachyzoites and prepared for TEM as described. In total, six tissue cysts of the N. caninum NC-5 strain, ®ve tissue cysts of the N. caninum NC-Liverpool strain, eight tissue cysts of T. gondii VEG strain and four tissue cysts of T. gondii ME-49 strain that had been ®xed in glutaraldehyde were examined by TEM in this study. 3. Results The tissue cysts, tachyzoites and bradyzoites of N. caninum and T. gondii were similar ultrastructurally, with the zoites containing all the organelles and inclusion bodies characteristically found in coccidian parasites including two apical rings, two polar rings, a conoid, a pellicle consisting of a plasmalemma and an inner membrane complex, 22 subpellicular microtubules, micronemes, rhoptries, mitochondria, a nucleus, Golgi complex, plastid (also called Golgi adjunct), ribosomes, polysomes, dense granules, amylopectin
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granules, lipid bodies, vesicles, endoplasmic reticulum, micropores, and a posterior pore (Figs. 1±11). Table 1 gives a list of the major ultrastructural features of the tachyzoites, tissue cysts and bradyzoites of N. caninum and T. gondii. 3.1. Tachyzoites Tachyzoites of both species were within a host cell parasitophorous vacuole (PV) that contained a well-developed tubulovesicular membrane network (Figs. 1 and 2). In cells infected with T. gondii, several host cell mitochondria were closely associated with the parasitophorous vacuolar membrane (PVM) (Fig. 2), whereas few host cell mitochondria were next to the PV surrounding N. caninum tachyzoites. Tachyzoites of N. caninum were slightly larger than those of T. gondii, measuring 7.52 mm (n = 20) and 6.81.5±3 mm (n = 20), respectively. Tachyzoites of N. caninum had 6±16 rhoptries compared with 4±10 for T. gondii. The rhoptries of N. caninum were homogenously electron-dense and some tachyzoites had as many as 4±6 rhoptries located posterior to the zoite nucleus. The rhoptries of T. gondii were labyrinthine (spongy) and rarely located in the posterior of tachyzoites. Occasionally, in N. caninum one or two rhoptries were looped back on themselves (Fig. 1), whereas looped-back rhoptries were rarely seen in T. gondii tachyzoites. Micronemes were numerous in N. caninum, but few in T. gondii. Some of the micronemes of N. caninum were oriented perpendicularly to the zoite pellicle, whereas those of T. gondii were oriented haphazardly. Dense granules were scattered throughout the cytoplasm of both species, but were more numerous in the posterior end of N. caninum tachyzoites and in the anterior end of T. gondii tachyzoites. (Figs. 1 and 2). Tachyzoites of both species multiplied by endodyogeny in which two progeny were formed within a mother zoite (Fig. 2). Micropores were commonly observed in T. gondii, but they were rare in N. caninum. The plastids of both species were similar in appearance, consisting of a moderately electron-dense, ®ne-granular core surrounded by multiple
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Figs. 3 and 4. Transmission EMs of bradyzoites of N. caninum and T. gondii. Fig. 3. N. caninum NC-Liverpool. The nucleus (Nu) is located subterminally and the cytoplasm behind the nucleus contains micronemes (Mn), six dense granules (Dg) and a small dense granule (Sg). Tubules (Tu) project from the inner surface of the cyst wall (Cw) and vesicles (arrowheads) are scattered amongst the bradyzoites. Fig. 4. Bradyzoite of T. gondii VEG strain with its nucleus (Nu) occupying its posterior. One of the rhoptries (Rh) is looped-back with the base of its neck projecting posteriorly; from Dubey et al. [16]. Abbreviations: Am, amylopectin; Ap, plastid; Ce, centrioles; Co, conoid; Go, Golgi complex; Mi, mitochondrion; Nr, neck of rhoptry; Pp, posterior pore.
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Fig. 5. Transmission EM of micropore of T. gondii bradyzoite. Am, amylopectin; Ec, electron-dense collar of micropore; Im, inner membrane complex; Pl, plasmalemma; Sm, subpellicular microtubule.
membranes (Fig. 2). Plastids of N. caninum were surrounded by two, and occasionally as many as four, membranes, whereas those of T. gondii had four membranes. Plastids were always closely associated with the parasite nucleus, with the Golgi complex and with centrioles, and pinched into two during endodyogeny with one half being incorporated into each progeny (Fig. 2). 3.2. Tissue cysts Tissue cysts of N. caninum and of T. gondii were found intracellularly in brain neural cells (Figs. 6, 7, 10 and 11). The interior of the tissue cysts was not separated into compartments by septa. The tissue cysts of N. caninum were smaller than those of T. gondii, containing 20±100 bradyzoites compared with 50±500 bradyzoites for T. gondii (Figs. 6 and 11). Tissue cysts of both species contained several dividing zoites that were in various stages of producing progeny by endodyogeny. The primary cyst wall of both coccidians consisted of a PVM and a granular layer (Figs. 6±11). In N. caninum NC-5, the margin of the tissue cyst was irregular in outline and variable in thickness, measuring 1.9 mm (0.5±4 mm)
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(Fig. 6). The tissue cyst walls of both strains of T. gondii were smooth and less than 0.5 mm thick. The granular layer of N. caninum NC-5 and T. gondii VEG and ME-49 contained electron-dense granules and vesicles embedded in a ®nely granular matrix (Figs. 7 and 10). The vesicles were larger, more electron-dense and more numerous in N. caninum NC-5 cyst walls than in T. gondii VEG and ME-49. In both coccidians, the vesicles appeared to pinch o from indentations of the PVM (Fig. 8). Host-cell endoplasmic reticulum was situated immediately above the PVM and often protruded into the invaginations (Fig. 8). Tubular structures measuring 75±85 or 30±40 mm in thickness and 300±950 mm in length arose from the inner aspect of the cyst walls of N. caninum NC-5 and T. gondii VEG and ME-49 (Figs. 7 and 9), some of which appeared to be in direct contact with the plasmalemmae of bradyzoites. The interior of the tissue cysts of N. caninum NC-5 and T. gondii VEG and ME-49 consisted of a ®ne granular matrix containing tubules and vesicles that were 30±450 nm in diameter and ®lled with moderately electron-dense material. Compared with N. caninum NC-5, the tissue cysts of N. caninum NC-Liverpool, that had been isolated from host tissue by Percoll density gradient centrifugation, were smaller and had smooth and slightly thicker cyst walls (2.4 mm; 2±4.3 mm; n = 5) with fewer electron-dense vesicles in the granular layer (Figs. 8 and 9). No ultrastructural dierences were detected between the tissue cysts of T. gondii VEG and ME-49. 3.3. Bradyzoites The bradyzoites of N. caninum were slightly longer than those of T. gondii, measuring 8.12 mm (6.5±101.5±2.5 mm; n = 16) and 7.5 2.5 mm (6.5±8.52±3 mm; n = 20), respectively. Compared with tachyzoites, bradyzoites of both species had fewer rhoptries and more amylopectin granules. The rhoptries were homogenously electron-dense in bradyzoites of both species (Figs. 3 and 4); however, in young tissue cysts of T. gondii some of the bradyzoites had labyrinthine rhoptries. In some specimens, it was di-
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Figs. 6 and 7. Transmission EM of N. caninum tissue cysts. Fig. 6. Tissue cyst of N. caninum NC-5 showing approximately 30 bradyzoites (Br) surrounded by an irregularly shaped cyst wall (Cw). Fig. 7. High magni®cation of portion of cyst wall in Fig. 6, showing electron-dense vesicles (Ev) of various sizes embedded in the granular layer (Gl) and large (Tu) and small (Tu*) tubules projecting from the inner aspect of the cyst wall. Host cell endoplasmic reticulum with distended cisternae (arrowheads) is closely associated with the parasitophorous vacuolar membrane (Pm). From Dubey et al. [7].
cult to distinguish between rhoptries and dense granules. Most bradyzoites of T. gondii had one to three looped-back rhoptries; looped-back rhoptries were rare in N. caninum bradyzoites. Both had similar numbers of micronemes (>40) and rhoptries (6±12), but in many bradyzoites of N. caninum the micronemes were oriented perpendicularly to the zoite pellicle. The nucleus of N. caninum bradyzoites was located approximately 1.5 mm anterior to the posterior tip of the zoite, whereas the nucleus in T. gondii bradyzoites usually occupied the posterior end (Figs. 3 and 4). In N. caninum, micronemes, dense granules, small dense granules, amylopectin granules, endoplasmic reticulum, and occasionally mitochondria were located in the cytoplasm between the nucleus and the posterior tip. Occasionally,
micronemes and dense granules were located near the lateral margins of the bradyzoite nucleus in T. gondii, but rarely posterior to the nucleus. The small dense granules were absent in bradyzoites of T. gondii. Longitudinal sections of N. caninum NC-5 and NC-Liverpool bradyzoites showed four to eight small dense granules scattered randomly in the cytoplasm (Fig. 3). Compared with dense granules, the small dense granules were smaller (120±170 nm vs 275± 400 nm in diameter), more electron-dense than dense granules, and arose from the forming face of the Golgi complex. In both species, numerous vesicles, some of which were multiple-membrane bound or contained several smaller vesicles, arose from the maturation face of the Golgi complex (Figs. 3 and 4). Micropores were common in
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Figs. 8±10. Transmission EMs of tissue cysts. Fig. 8. Brain tissue cyst of N. caninum NC-Liverpool isolated by Percoll centrifugation showing approximately 17 bradyzoites. Fig. 9. Higher magni®cation of portion of cyst wall in Fig. 8 partially surrounded by segments of the parasitophorous vacuolar membrane (Pm), small tubules (Tu) projecting from the inner aspect of the cyst wall, and vesicles (Vs) of variable shapes and sizes within the cyst. Fig. 10. High magni®cation of the cyst wall of T. gondii. Note that the endoplasmic reticulum in the host cell cytoplasm (Hc) is closely associated with the parasitophorous vacuolar membrane (Pm). The cisterna of the host cell endoplasmic reticulum is distended near invaginations (arrows) of the parasitophorous vacuole. The host cell endoplasmic reticulum has ribosomes attached to the cytoplasmic side, but the endoplasmic reticular membrane adjacent to the parasitophorous vacuolar membrane lacks ribosomes.
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Fig. 11. Transmission EM of a tissue cyst of the VEG strain of T. gondii showing the relatively thin cyst wall, a dividing zoite (*) and numerous bradyzoites (Br).
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Table 1 Comparative ultrastructure of tachyzoites, tissue cysts, and bradyzoites of Neospora caninum and Toxoplasma gondii Parasite stage/structure Tachyzoite Anterior rhoptriesa Posterior rhoptriesb Looped-back rhoptries Anterior micronemes Posterior micronemes Anterior dense granules Posterior dense granules Micropores Tissue cyst wall Thickness Contour Bradyzoites Anterior rhoptries Posterior rhoptries Looped-back rhoptries Anterior micronemes Posterior micronemes Anterior dense granules Posterior dense granules Small dense granules Micropores
N. caninum
T. gondii
Electron-dense, 6±16 Electron-dense Electron-dense, 1±2 Many Few Several Several Rare
Labyrinthine, 4±10 Rare Rare Few Rare Several Few Common
0.5±4 mm Irregular
R0.5 mm Smooth
Electron-dense, 6±12 Electron-dense, rare None Many Few Several Several 4±8 Rare
Labyrinthine in young cysts, electron-dense in mature cysts, 6±8 None Electron-dense, 1±3 Many Rare Several Rare None Common
a
Rhoptries located anterior to the parasite nucleus. Rhoptries located posterior to the parasite nucleus.
b
bradyzoites of T. gondii (Fig. 5), but rare in those of N. caninum. Plastids of both species were similar to those described for tachyzoites. There were no ultrastructural dierences between the bradyzoites of the NC-Liverpool and the NC-5 strains of N. caninum, nor between the bradyzoites of the VEG and ME-49 strains of T. gondii. 4. Discussion Limited information is available concerning the ultrastructure of the tissue cysts and bradyzoites of N. caninum. The main reason is the paucity of the tissue cysts. Compared with T. gondii, tissue cysts of N. caninum are few in number in naturally infected animals, and there is no consistent method of producing them in experimentally infected animals. Neospora tissue cysts have not been reported in cell cultures. Therefore, there are only a few reports of the ultrastructure
of tissue cysts in dogs [7, 10±12] and in cattle [13, 14]. All of these reports were based on formalin-®xed material, except one tissue cyst described by Speer and Dubey [12]. In the present study, the ultrastructure of 11 glutaraldehyde-®xed tissue cysts, six from a naturally infected dog and ®ve tissue cysts separated from the brain of an experimentally infected mouse, were found to be ultrastructurally similar. Although the characteristics of the tachyzoites and bradyzoites of N. caninum and T. gondii are basically similar ultrastructurally, there are several features that can be used to dierentiate the two coccidians. For instance, the rhoptries of N. caninum tachyzoites are homogenously electrondense, whereas those of T. gondii are labyrinthine (spongy) (present study, [15, 16]). In N. caninum, some of the micronemes are oriented perpendicular to the pellicle, whereas in T. gondii the micronemes are arranged haphazardly. The tachyzoites (merozoites) of Hammondia heydorni are closer in appearance ultrastructurally to N. caninum than
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to T. gondii. In N. caninum and H. heydorni the cytoplasm posterior to the nucleus contains more dense granules, rhoptries and micronemes than do tachyzoites of T. gondii [12, 17]. More ultrastructural dierences exist between the bradyzoites of N. caninum and T. gondii. The rhoptries of the bradyzoites of T. gondii are labyrinthine in young cysts and electron dense in older cysts (present study, [18, 19]). In T. gondii, there are one to three looped-back rhoptries per bradyzoite (present study, [16, 19]), but there are no looped-back rhoptries in N. caninum. In contrast to bradyzoites of T. gondii, some of the micronemes in bradyzoites of N. caninum are oriented perpendicularly to the zoite pellicle. One of the major dierences is the location of the bradyzoite nucleus. In N. caninum, the nucleus is located subterminally, whereas in T. gondii the nucleus occupies the posterior end. Because of the nuclear location in N. caninum, the cytoplasm behind the nucleus contains several organelles and inclusion bodies such as micronemes, occasionally rhoptries, dense granules, amylopectin granules, small dense granules, endoplasmic reticulum and mitochondria. The small dense granules that appeared to be formed by the forming face of the Golgi complex in bradyzoites of N. caninum were not present in T. gondii bradyzoites. Small dense granules have not been reported for other coccidians and their signi®cance is not known. The bradyzoites of H. hammondi are essentially identical ultrastructurally to those of T. gondii, except that the former are substantially smaller [20], measuring 4±51.2 mm compared with 7±81.5±2.5 mm for T. gondii (present study, [20]). In general, the tissue cyst wall of N. caninum NC-5 is thicker (0.5±4 mm) and more irregular in shape than that of T. gondii (0.5 mm) (present study, [16±21]). In the present study, tissue cysts of the NC-Liverpool strain of N. caninum had thicker (up to 4.3 mm) and smoother walls with fewer vesicles embedded in the granular layer than did the NC-5 strain. This was probably due to the eects on the cyst walls of the Percoll isolation procedure. In the present study, tissue cysts of N. caninum were structurally similar to those described
previously [3, 10, 11, 14]. However, Jardine [14] reported that bradyzoites in tissue cysts from one dog were separated into compartments by branching septa. Septa were not observed in any of the tissue cysts we examined. The septa observed by Jardine [14] may either be artifacts or the cysts may belong to another parasite, because septa are lacking in the tissue cysts of N. caninum [10, 14]. In a previous study, Kohler et al. [22] proposed the name `apicoplast' for the multiple membrane-bound structures found in tachyzoites of T. gondii. A phylogenetic analysis of a plastid gene (TUfA) placed this organellar genome with cyanobacteria and plastids, especially green algal plastids [22]. Prior to Kohler's report, these structures, which had been seen for many years in various species of Eimeria and Sarcocystis as well as T. gondii, had been most commonly referred to as `Golgi adjuncts' [23]. The term Golgi adjunct was used because they were always closely associated with Golgi complexes. In a previous study on Sarcocystis falcatula [24], we found a putative plastid closely associated with nuclear spindles and centrioles, indicating that plastids may play a role in nuclear division. We found in the present study that tachyzoites and bradyzoites of N. caninum also appear to contain a putative plastid consisting of a granular matrix surrounded by two, and occasionally as many as four, membranes. The plastid of T. gondii is limited by four membranes [22]. Further study will be needed to determine if these structures are analogous to the plastid of T. gondii tachyzoites.
Acknowledgements This study was funded in part by grants from the Montana State University Agricultural Experiment Station and the US Department of Agriculture.
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Comparative ultrastructure of tachyzoites, bradyzoites, and tissue cysts of Neospora caninum and Toxoplasma gondii C.A. Speer a, *, J.P. Dubey b, M.M. McAllister c, J.A. Blixt a a
Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717-3610, USA United States Department of Agriculture, Agriculture Research Service, Livestock and Poultry Sciences Institute, Parasite Biology and Epidemiology Laboratory, BARC-East, Bldg. 1001, Beltsville, MD 20705-2350, USA c Department of Veterinary Pathology, University of Illinois, 2522 VMBSB, 2001 S. Lincoln Avenue, Urbana, IL 61802, USA
b
Received 29 April 1999; received in revised form 16 July 1999; accepted 16 July 1999
Abstract The ultrastructure of tachyzoites, bradyzoites and tissue cysts of the NC-1, NC-5 and NC-Liverpool strains of Neospora caninum are reviewed and compared with those of the VEG and ME-49 strains of Toxoplasma gondii. While each stage of N. caninum and T. gondii shared many ultrastructural characteristics, each parasite stage also had certain features or organelles that could be used to distinguish the two parasites. Some of the most prominent ultrastructural dierences occurred in the number, appearance and location of rhoptries, looped-back rhoptries, micronemes, dense granules, small dense granules and micropores. The tissue cysts of both parasites were also basically similar, being surrounded by a cyst wall and not compartmentalised by septa. The cyst wall of N. caninum was irregular and substantially thicker, 0.5±4 mm, than those of T. gondii which were smooth and 0.5 mm thick. # 1999 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Bradyzoites; Neospora caninum; Tachyzoites; Tissue cysts; Toxoplasma gondii; Ultrastructure
1. Introduction
2. Materials and methods
The tachyzoites, bradyzoites and tissue cysts of Neospora caninum and Toxoplasma gondii appear essentially identical by standard light microscopy. These parasites can, however, be dierentiated on the basis of certain ultrastructural features [1± 3]. In this paper, we review and compare the ultrastructure of the tachyzoites, bradyzoites and tissue cysts of N. caninum with those of T. gondii.
Tachyzoites of the NC-1 strain of N. caninum [4] were derived from cultured bovine monocytes (M617) [5] and bovine pulmonary artery endothelial cells (American Type Culture Collection). Tachyzoites of the VEG and ME-49 strains of T. gondii [6] were obtained from cultured M617 cells, CPA cells (ATCC), or the peritoneum of a Swiss Webster mouse that had been inoculated 6 days earlier with tachyzoites. Specimens were ®xed in 3% glutaraldehyde in Millonig's phosphate buer or modi®ed
* Corresponding author. Tel.: +1-406-994-6389; fax: +1406-994-6878.
0020-7519/99/$20.00 # 1999 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 0 - 7 5 1 9 ( 9 9 ) 0 0 1 3 2 - 0
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Figs. 1 and 2. Transmission EM of tachyzoites of N. caninum and T. gondii. Fig. 1. Tachyzoite of N. caninum NC-5 in a parasitophorous vacuole (PV) within a cultured M617 cell, showing various organelles and inclusion bodies. Some of the micronemes (Mn) are oriented perpendicularly to the zoite pellicle and the rhoptries are uniformly electron-dense, one of which (Rh) is oriented with its neck projecting posteriorly. Approximately 10 dense granules (Dg) are situated posterior and three anterior to the nucleus. Fig. 2. Tachyzoite of T. gondii VEG strain in a parasitophorous vacuole (PV) within a mouse peritoneal macrophage, showing labyrinthine rhoptries (Rh) and an early stage of endodyogeny in which a progeny zoite, consisting of a plastid (Ap*), Golgi complex (Go*), inner membrane complex (Im*), and rhoptries (Rh*), is developing internally within the mother zoite. Several host-cell mitochondria (*) are situated close to the parasitophorous vacuole. Abbreviations: Am, amylopectin; Ar 1, 2, apical rings 1 and 2; Co, conoid; Go, Golgi complex; Hc, host cell cytoplasm; Im, inner membrane complex of pellicle; Lb, lipid body; Mi, mitochondrion; Mn, microneme; No, nucleolus; Nu, nucleus; Pl, plasmalemma; Pr 1, 2, polar rings 1 and 2; Sm, subpellicular microtubule; Tm, tubulovesicular membrane network.
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Karnovsky's ®xative, rinsed in Millonig's phosphate buer or cacodylate buer, post-®xed in 1% osmium tetroxide, rinsed in buer, dehydrated in ethanol, and embedded in Spurr's medium. Some of the specimens were prestained with 1% uranyl acetate and 1% phosphotungstic acid in 70% ethanol overnight, prior to complete dehydration and embedding. Before examination in a JEOL 100CX TEM, ultrathin sections of prestained specimens were stained with lead citrate; non-prestained specimens were stained with uranyl acetate and lead citrate. Tissue cysts of N. caninum and T. gondii were obtained from infected brains and had been ®xed in Karnovsky's ®xative. Six tissue cysts were obtained from parts of the brain of a dog with NC-isolate [7]. Five N. caninum NC-Liverpool tissue cysts studied had been freed from the brains of infected mice using Percoll [8]. The mice had been inoculated with tachyzoites of the NC-Liverpool strain of N. caninum [9]. Tissue cysts were washed free of Percoll, and prepared for TEM as described above. Tissue cysts of the VEG or ME-49 strain of T. gondii were obtained from Swiss Webster mice that had been inoculated 7±9 months earlier with tachyzoites and prepared for TEM as described. In total, six tissue cysts of the N. caninum NC-5 strain, ®ve tissue cysts of the N. caninum NC-Liverpool strain, eight tissue cysts of T. gondii VEG strain and four tissue cysts of T. gondii ME-49 strain that had been ®xed in glutaraldehyde were examined by TEM in this study. 3. Results The tissue cysts, tachyzoites and bradyzoites of N. caninum and T. gondii were similar ultrastructurally, with the zoites containing all the organelles and inclusion bodies characteristically found in coccidian parasites including two apical rings, two polar rings, a conoid, a pellicle consisting of a plasmalemma and an inner membrane complex, 22 subpellicular microtubules, micronemes, rhoptries, mitochondria, a nucleus, Golgi complex, plastid (also called Golgi adjunct), ribosomes, polysomes, dense granules, amylopectin
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granules, lipid bodies, vesicles, endoplasmic reticulum, micropores, and a posterior pore (Figs. 1±11). Table 1 gives a list of the major ultrastructural features of the tachyzoites, tissue cysts and bradyzoites of N. caninum and T. gondii. 3.1. Tachyzoites Tachyzoites of both species were within a host cell parasitophorous vacuole (PV) that contained a well-developed tubulovesicular membrane network (Figs. 1 and 2). In cells infected with T. gondii, several host cell mitochondria were closely associated with the parasitophorous vacuolar membrane (PVM) (Fig. 2), whereas few host cell mitochondria were next to the PV surrounding N. caninum tachyzoites. Tachyzoites of N. caninum were slightly larger than those of T. gondii, measuring 7.52 mm (n = 20) and 6.81.5±3 mm (n = 20), respectively. Tachyzoites of N. caninum had 6±16 rhoptries compared with 4±10 for T. gondii. The rhoptries of N. caninum were homogenously electron-dense and some tachyzoites had as many as 4±6 rhoptries located posterior to the zoite nucleus. The rhoptries of T. gondii were labyrinthine (spongy) and rarely located in the posterior of tachyzoites. Occasionally, in N. caninum one or two rhoptries were looped back on themselves (Fig. 1), whereas looped-back rhoptries were rarely seen in T. gondii tachyzoites. Micronemes were numerous in N. caninum, but few in T. gondii. Some of the micronemes of N. caninum were oriented perpendicularly to the zoite pellicle, whereas those of T. gondii were oriented haphazardly. Dense granules were scattered throughout the cytoplasm of both species, but were more numerous in the posterior end of N. caninum tachyzoites and in the anterior end of T. gondii tachyzoites. (Figs. 1 and 2). Tachyzoites of both species multiplied by endodyogeny in which two progeny were formed within a mother zoite (Fig. 2). Micropores were commonly observed in T. gondii, but they were rare in N. caninum. The plastids of both species were similar in appearance, consisting of a moderately electron-dense, ®ne-granular core surrounded by multiple
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Figs. 3 and 4. Transmission EMs of bradyzoites of N. caninum and T. gondii. Fig. 3. N. caninum NC-Liverpool. The nucleus (Nu) is located subterminally and the cytoplasm behind the nucleus contains micronemes (Mn), six dense granules (Dg) and a small dense granule (Sg). Tubules (Tu) project from the inner surface of the cyst wall (Cw) and vesicles (arrowheads) are scattered amongst the bradyzoites. Fig. 4. Bradyzoite of T. gondii VEG strain with its nucleus (Nu) occupying its posterior. One of the rhoptries (Rh) is looped-back with the base of its neck projecting posteriorly; from Dubey et al. [16]. Abbreviations: Am, amylopectin; Ap, plastid; Ce, centrioles; Co, conoid; Go, Golgi complex; Mi, mitochondrion; Nr, neck of rhoptry; Pp, posterior pore.
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Fig. 5. Transmission EM of micropore of T. gondii bradyzoite. Am, amylopectin; Ec, electron-dense collar of micropore; Im, inner membrane complex; Pl, plasmalemma; Sm, subpellicular microtubule.
membranes (Fig. 2). Plastids of N. caninum were surrounded by two, and occasionally as many as four, membranes, whereas those of T. gondii had four membranes. Plastids were always closely associated with the parasite nucleus, with the Golgi complex and with centrioles, and pinched into two during endodyogeny with one half being incorporated into each progeny (Fig. 2). 3.2. Tissue cysts Tissue cysts of N. caninum and of T. gondii were found intracellularly in brain neural cells (Figs. 6, 7, 10 and 11). The interior of the tissue cysts was not separated into compartments by septa. The tissue cysts of N. caninum were smaller than those of T. gondii, containing 20±100 bradyzoites compared with 50±500 bradyzoites for T. gondii (Figs. 6 and 11). Tissue cysts of both species contained several dividing zoites that were in various stages of producing progeny by endodyogeny. The primary cyst wall of both coccidians consisted of a PVM and a granular layer (Figs. 6±11). In N. caninum NC-5, the margin of the tissue cyst was irregular in outline and variable in thickness, measuring 1.9 mm (0.5±4 mm)
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(Fig. 6). The tissue cyst walls of both strains of T. gondii were smooth and less than 0.5 mm thick. The granular layer of N. caninum NC-5 and T. gondii VEG and ME-49 contained electron-dense granules and vesicles embedded in a ®nely granular matrix (Figs. 7 and 10). The vesicles were larger, more electron-dense and more numerous in N. caninum NC-5 cyst walls than in T. gondii VEG and ME-49. In both coccidians, the vesicles appeared to pinch o from indentations of the PVM (Fig. 8). Host-cell endoplasmic reticulum was situated immediately above the PVM and often protruded into the invaginations (Fig. 8). Tubular structures measuring 75±85 or 30±40 mm in thickness and 300±950 mm in length arose from the inner aspect of the cyst walls of N. caninum NC-5 and T. gondii VEG and ME-49 (Figs. 7 and 9), some of which appeared to be in direct contact with the plasmalemmae of bradyzoites. The interior of the tissue cysts of N. caninum NC-5 and T. gondii VEG and ME-49 consisted of a ®ne granular matrix containing tubules and vesicles that were 30±450 nm in diameter and ®lled with moderately electron-dense material. Compared with N. caninum NC-5, the tissue cysts of N. caninum NC-Liverpool, that had been isolated from host tissue by Percoll density gradient centrifugation, were smaller and had smooth and slightly thicker cyst walls (2.4 mm; 2±4.3 mm; n = 5) with fewer electron-dense vesicles in the granular layer (Figs. 8 and 9). No ultrastructural dierences were detected between the tissue cysts of T. gondii VEG and ME-49. 3.3. Bradyzoites The bradyzoites of N. caninum were slightly longer than those of T. gondii, measuring 8.12 mm (6.5±101.5±2.5 mm; n = 16) and 7.5 2.5 mm (6.5±8.52±3 mm; n = 20), respectively. Compared with tachyzoites, bradyzoites of both species had fewer rhoptries and more amylopectin granules. The rhoptries were homogenously electron-dense in bradyzoites of both species (Figs. 3 and 4); however, in young tissue cysts of T. gondii some of the bradyzoites had labyrinthine rhoptries. In some specimens, it was di-
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Figs. 6 and 7. Transmission EM of N. caninum tissue cysts. Fig. 6. Tissue cyst of N. caninum NC-5 showing approximately 30 bradyzoites (Br) surrounded by an irregularly shaped cyst wall (Cw). Fig. 7. High magni®cation of portion of cyst wall in Fig. 6, showing electron-dense vesicles (Ev) of various sizes embedded in the granular layer (Gl) and large (Tu) and small (Tu*) tubules projecting from the inner aspect of the cyst wall. Host cell endoplasmic reticulum with distended cisternae (arrowheads) is closely associated with the parasitophorous vacuolar membrane (Pm). From Dubey et al. [7].
cult to distinguish between rhoptries and dense granules. Most bradyzoites of T. gondii had one to three looped-back rhoptries; looped-back rhoptries were rare in N. caninum bradyzoites. Both had similar numbers of micronemes (>40) and rhoptries (6±12), but in many bradyzoites of N. caninum the micronemes were oriented perpendicularly to the zoite pellicle. The nucleus of N. caninum bradyzoites was located approximately 1.5 mm anterior to the posterior tip of the zoite, whereas the nucleus in T. gondii bradyzoites usually occupied the posterior end (Figs. 3 and 4). In N. caninum, micronemes, dense granules, small dense granules, amylopectin granules, endoplasmic reticulum, and occasionally mitochondria were located in the cytoplasm between the nucleus and the posterior tip. Occasionally,
micronemes and dense granules were located near the lateral margins of the bradyzoite nucleus in T. gondii, but rarely posterior to the nucleus. The small dense granules were absent in bradyzoites of T. gondii. Longitudinal sections of N. caninum NC-5 and NC-Liverpool bradyzoites showed four to eight small dense granules scattered randomly in the cytoplasm (Fig. 3). Compared with dense granules, the small dense granules were smaller (120±170 nm vs 275± 400 nm in diameter), more electron-dense than dense granules, and arose from the forming face of the Golgi complex. In both species, numerous vesicles, some of which were multiple-membrane bound or contained several smaller vesicles, arose from the maturation face of the Golgi complex (Figs. 3 and 4). Micropores were common in
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Figs. 8±10. Transmission EMs of tissue cysts. Fig. 8. Brain tissue cyst of N. caninum NC-Liverpool isolated by Percoll centrifugation showing approximately 17 bradyzoites. Fig. 9. Higher magni®cation of portion of cyst wall in Fig. 8 partially surrounded by segments of the parasitophorous vacuolar membrane (Pm), small tubules (Tu) projecting from the inner aspect of the cyst wall, and vesicles (Vs) of variable shapes and sizes within the cyst. Fig. 10. High magni®cation of the cyst wall of T. gondii. Note that the endoplasmic reticulum in the host cell cytoplasm (Hc) is closely associated with the parasitophorous vacuolar membrane (Pm). The cisterna of the host cell endoplasmic reticulum is distended near invaginations (arrows) of the parasitophorous vacuole. The host cell endoplasmic reticulum has ribosomes attached to the cytoplasmic side, but the endoplasmic reticular membrane adjacent to the parasitophorous vacuolar membrane lacks ribosomes.
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Fig. 11. Transmission EM of a tissue cyst of the VEG strain of T. gondii showing the relatively thin cyst wall, a dividing zoite (*) and numerous bradyzoites (Br).
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Table 1 Comparative ultrastructure of tachyzoites, tissue cysts, and bradyzoites of Neospora caninum and Toxoplasma gondii Parasite stage/structure Tachyzoite Anterior rhoptriesa Posterior rhoptriesb Looped-back rhoptries Anterior micronemes Posterior micronemes Anterior dense granules Posterior dense granules Micropores Tissue cyst wall Thickness Contour Bradyzoites Anterior rhoptries Posterior rhoptries Looped-back rhoptries Anterior micronemes Posterior micronemes Anterior dense granules Posterior dense granules Small dense granules Micropores
N. caninum
T. gondii
Electron-dense, 6±16 Electron-dense Electron-dense, 1±2 Many Few Several Several Rare
Labyrinthine, 4±10 Rare Rare Few Rare Several Few Common
0.5±4 mm Irregular
R0.5 mm Smooth
Electron-dense, 6±12 Electron-dense, rare None Many Few Several Several 4±8 Rare
Labyrinthine in young cysts, electron-dense in mature cysts, 6±8 None Electron-dense, 1±3 Many Rare Several Rare None Common
a
Rhoptries located anterior to the parasite nucleus. Rhoptries located posterior to the parasite nucleus.
b
bradyzoites of T. gondii (Fig. 5), but rare in those of N. caninum. Plastids of both species were similar to those described for tachyzoites. There were no ultrastructural dierences between the bradyzoites of the NC-Liverpool and the NC-5 strains of N. caninum, nor between the bradyzoites of the VEG and ME-49 strains of T. gondii. 4. Discussion Limited information is available concerning the ultrastructure of the tissue cysts and bradyzoites of N. caninum. The main reason is the paucity of the tissue cysts. Compared with T. gondii, tissue cysts of N. caninum are few in number in naturally infected animals, and there is no consistent method of producing them in experimentally infected animals. Neospora tissue cysts have not been reported in cell cultures. Therefore, there are only a few reports of the ultrastructure
of tissue cysts in dogs [7, 10±12] and in cattle [13, 14]. All of these reports were based on formalin-®xed material, except one tissue cyst described by Speer and Dubey [12]. In the present study, the ultrastructure of 11 glutaraldehyde-®xed tissue cysts, six from a naturally infected dog and ®ve tissue cysts separated from the brain of an experimentally infected mouse, were found to be ultrastructurally similar. Although the characteristics of the tachyzoites and bradyzoites of N. caninum and T. gondii are basically similar ultrastructurally, there are several features that can be used to dierentiate the two coccidians. For instance, the rhoptries of N. caninum tachyzoites are homogenously electrondense, whereas those of T. gondii are labyrinthine (spongy) (present study, [15, 16]). In N. caninum, some of the micronemes are oriented perpendicular to the pellicle, whereas in T. gondii the micronemes are arranged haphazardly. The tachyzoites (merozoites) of Hammondia heydorni are closer in appearance ultrastructurally to N. caninum than
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to T. gondii. In N. caninum and H. heydorni the cytoplasm posterior to the nucleus contains more dense granules, rhoptries and micronemes than do tachyzoites of T. gondii [12, 17]. More ultrastructural dierences exist between the bradyzoites of N. caninum and T. gondii. The rhoptries of the bradyzoites of T. gondii are labyrinthine in young cysts and electron dense in older cysts (present study, [18, 19]). In T. gondii, there are one to three looped-back rhoptries per bradyzoite (present study, [16, 19]), but there are no looped-back rhoptries in N. caninum. In contrast to bradyzoites of T. gondii, some of the micronemes in bradyzoites of N. caninum are oriented perpendicularly to the zoite pellicle. One of the major dierences is the location of the bradyzoite nucleus. In N. caninum, the nucleus is located subterminally, whereas in T. gondii the nucleus occupies the posterior end. Because of the nuclear location in N. caninum, the cytoplasm behind the nucleus contains several organelles and inclusion bodies such as micronemes, occasionally rhoptries, dense granules, amylopectin granules, small dense granules, endoplasmic reticulum and mitochondria. The small dense granules that appeared to be formed by the forming face of the Golgi complex in bradyzoites of N. caninum were not present in T. gondii bradyzoites. Small dense granules have not been reported for other coccidians and their signi®cance is not known. The bradyzoites of H. hammondi are essentially identical ultrastructurally to those of T. gondii, except that the former are substantially smaller [20], measuring 4±51.2 mm compared with 7±81.5±2.5 mm for T. gondii (present study, [20]). In general, the tissue cyst wall of N. caninum NC-5 is thicker (0.5±4 mm) and more irregular in shape than that of T. gondii (0.5 mm) (present study, [16±21]). In the present study, tissue cysts of the NC-Liverpool strain of N. caninum had thicker (up to 4.3 mm) and smoother walls with fewer vesicles embedded in the granular layer than did the NC-5 strain. This was probably due to the eects on the cyst walls of the Percoll isolation procedure. In the present study, tissue cysts of N. caninum were structurally similar to those described
previously [3, 10, 11, 14]. However, Jardine [14] reported that bradyzoites in tissue cysts from one dog were separated into compartments by branching septa. Septa were not observed in any of the tissue cysts we examined. The septa observed by Jardine [14] may either be artifacts or the cysts may belong to another parasite, because septa are lacking in the tissue cysts of N. caninum [10, 14]. In a previous study, Kohler et al. [22] proposed the name `apicoplast' for the multiple membrane-bound structures found in tachyzoites of T. gondii. A phylogenetic analysis of a plastid gene (TUfA) placed this organellar genome with cyanobacteria and plastids, especially green algal plastids [22]. Prior to Kohler's report, these structures, which had been seen for many years in various species of Eimeria and Sarcocystis as well as T. gondii, had been most commonly referred to as `Golgi adjuncts' [23]. The term Golgi adjunct was used because they were always closely associated with Golgi complexes. In a previous study on Sarcocystis falcatula [24], we found a putative plastid closely associated with nuclear spindles and centrioles, indicating that plastids may play a role in nuclear division. We found in the present study that tachyzoites and bradyzoites of N. caninum also appear to contain a putative plastid consisting of a granular matrix surrounded by two, and occasionally as many as four, membranes. The plastid of T. gondii is limited by four membranes [22]. Further study will be needed to determine if these structures are analogous to the plastid of T. gondii tachyzoites.
Acknowledgements This study was funded in part by grants from the Montana State University Agricultural Experiment Station and the US Department of Agriculture.
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