Ultrastructure of Besnoitia besnoiti tissue cysts and bradyzoites

Besnoitia besnoiti is an economically important tissue cyst-forming apicomplexan of cattle in Africa and Israel. Tissue cysts and bradyzoites of B. besnoiti from the skin of a naturally infected bull were studied by transmission electron microscopy. Tissue cysts enclosed host cell and bradyzoites. Bradyzoites were 6-7.5 x 1.9-2.3 microm in size and contained organelles found in coccidian merozoites including numerous micronemes, rhoptries, amylopectin granules, and a posteriorly located nucleus. Enigmatic bodies, characteristically found in Besnoitia sp. bradyzoites, were not observed. Therefore, enigmatic bodies should be removed as a generic character of the bradyzoites of Besnoitia species.     

Redescription of Besnoitia tarandi (Protozoa: Apicomplexa) from the reindeer (Rangifer tarandus)

Besnoitia tarandi tissue cysts were found in naturally-infected reindeer (Rangifer tarandus) from Finland. Infectivity of its tissue cysts, bradyzoites, and tachyzoites to animals and cell culture was studied. The bradyzoites and tissue cysts were not infectious to out-bred mice, rabbits or gerbils. When fed tissue cysts, neither cats nor dogs excreted oocysts. However, the parasite was lethal to interferon-gamma gene knock out mice irrespective of the route of inoculation. The parasite was grown successfully in African Green Monkey cells from tissues of two reindeer for the first time. Non-dividing, uninucleate tachyzoites from smears from cell cultures were 5.6 x 1.4 microm (4.5-7.4 x 1.0-1.9, n=50) in size. Longitudinally-cut bradyzoites in tissue sections measured 7.4 x 1.3 microm (6.5-7.8 x 1.0-1.6, n=30). Ultrastructurally, tachyzoites and bradyzoites were similar to those in other Besnoitia species, and in particular to parasites described from cattle (Besnoitia besnoiti) and equids (Besnoitia bennetti) in that their bradyzoites lacked enigmatic bodies. Based on comparative analysis of three portions of nuclear ribosomal DNA (the small and large subunits and the first internal transcribed spacer) B. tarandi was found to be more closely related to the other congeners described from ungulates. The parasite was formally redescribed and specimens deposited in the US National Parasite Collection.     

Redescription of Besnoitia bennetti (Protozoa: Apicomplexa) from the donkey (Equus asinus)

Besnoitia bennetti tissue cysts were found in four naturally-infected donkeys (Equus asinus) from the USA. Infectivity of its bradyzoites and tachyzoites to animals and cell culture was studied. The bradyzoites were not infectious to out-bred Swiss Webster mice, rabbits or gerbils. When fed tissue cysts, cats did not excrete oocysts. However, the parasite was infectious to interferon-gamma gene knock out mice. The parasite from tissues of two donkeys was grown successfully in bovine monocyte monolayers for the first time. Non-dividing, uninucleate tachyzoites were approximately 6 x 1.5 microm in size. Longitudinally-cut bradyzoites in tissue sections measured 8.7 x 1.9 microm. Ultrastructurally, tachyzoites and bradyzoites were similar to those in other Besnoitia species, and in particular to parasites described from cattle (Besnoitia besnoiti) and reindeer (Besnoitia tarandi), in that their bradyzoites lacked enigmatic bodies. Based on comparative analysis of three portions of nuclear ribosomal DNA (the small and large subunits and the first internal transcribed spacer) B. bennetti was found to be more closely related to the other congeners described from ungulates. The parasite was formally redescribed and specimens deposited in the US National Parasite Collections.     

Biological and molecular characterization of Besnoitia akodoni n.sp. (Protozoa: Apicomplexa) from the rodent Akodon montensis in Brazil

The diversity among coccidian parasites of the genus Besnoitia is incompletely known. Of the eight currently described members of the genus, only B. jellisoni is known to parasitize a rodent host. Here, we propose a new name, Besnoitia akodoni, for the species initially isolated form the rodent Akodon montensis in Brazil. The tissue cysts of B. akodoni were up to 442 microm in diameter and bradyzoites were 8.4 x 1.4 microm in size. The bradyzoites contained enigmatic bodies, micronemes and rhoptries. Tachyzoites were 5.8 x 1.5 microm in size and they could be grown in vitro in bovine monocytes and African Green monkey cells where they divided by endodyogeny. Besnoitia akodoni was infective to laboratory-raised mice (Mus musculus) and gerbils (Meriones unguiculatus) but not to cats (Felis catus). Comparison of the conserved sequences of the small subunit rDNA clearly established the close relationship of B. akodoni with other members of the genus. However, sequences of the more variable first internal transcribed spacer portion of the ribosomal DNA repeat support its differentiation from the other species of the genus.     

Establishment of Besnoitia darlingi from opossums (Didelphis virginiana) in experimental intermediate and definitive hosts,propagation in cell culture,and description of ultrastructural and genetic characteristics

Besnoitia darlingi from naturally infected opossums (Didelphis virginiana) from Mississippi, USA, was propagated experimentally in mice, cats, and cell culture and was characterised according to ultrastructural, genetic, and life-history characteristics. Cats fed tissue cysts from opossums shed oocysts with a prepatent period of nine or 11 days. Oocysts, bradyzoites, or tachyzoites were infective to outbred and interferon-gamma gene knockout mice. Tachyzoites were successfully cultivated and maintained in vitro in bovine monocytes and African green monkey cells and revived after an 18-month storage in liquid nitrogen. Schizonts were seen in the small intestinal lamina propria of cats fed experimentally-infected mouse tissues. These schizonts measured up to 45 x 25 microm and contained many merozoites. A few schizonts were present in mesenteric lymph nodes and livers of cats fed tissue cysts. Ultrastructurally, tachyzoites and bradyzoites of B. darlingi were similar to other species of Besnoitia. A close relationship to B. besnoiti and an even closer relationship to B. jellisoni was indicated for B. darlingi on the basis of the small subunit and ITS-1 portions of nuclear ribosomal DNA.     

Comparative ultrastructure of tachyzoites, bradyzoites, and tissue cysts of Neospora caninum and Toxoplasma gondii

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 differences 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 microm, than those of T. gondii which were smooth and 0.5 microm thick.     

Ultrastructure of Tachyzoites, Bradyzoites and Tissue Cysts of Neospora caninum

. Dividing tachyzoites of Neospora caninum were 4x3 μm and had ultrastructural characteristics typical for the cyst-forming coccidia. Unusual ultrastructural characteristics of fully-formed tachyzoites included no micropores, 8–12 anterior and 4–6 posterior rhoptries, and a few posterior micronemes. Most tachyzoites were located free in the host cell cytoplasm; only a few occurred within a parasitophorous vacuole. Parasite multiplication appeared to be rapid because most organisms were in various stages of endodyogeny. Neural tissue cysts of N. caninum were 24.3 × 19.2 μm and contained 50–200 bradyzoites (7.3 × 1.5 μm), which lacked micropores. The cyst wall was 0.74–1.12 μm thick and consisted of the primary cyst wall (the parasitophorous vacuole membrane) and a thick granular layer with electron-dense vesicles.     

Ultrastructure of tachyzoites, bradyzoites and tissue cysts of Neospora caninum

Dividing tachyzoites of Neospora caninum were 4 x 3 microns and had ultrastructural characteristics typical for the cyst-forming coccidia. Unusual ultrastructural characteristics of fully-formed tachyzoites included no micropores, 8-12 anterior and 4-6 posterior rhoptries, and a few posterior micronemes. Most tachyzoites were located free in the host cell cytoplasm; only a few occurred within a parasitophorous vacuole. Parasite multiplication appeared to be rapid because most organisms were in various stages of endodyogeny. Neural tissue cysts of N. caninum were 24.3 x 19.2 microns and contained 50-200 bradyzoites (7.3 x 1.5 microns), which lacked micropores. The cyst wall was 0.74-1.12 microns thick and consisted of the primary cyst wall (the parasitophorous vacuole membrane) and a thick granular layer with electron-dense vesicles.     

Feline Toxoplasmosis from Acutely Infected Mice and the Development of Toxoplasma Cysts*

SYNOPSIS. The development of Toxoplasma cysts was studied in mice inoculated with tachyzoites by several routes. After 1–30 days of infection, murine tissues were examined microscopically, and portions or whole carcasses were fed to mice and cats. The feces of the cats were examined for oocyst shedding. Cyst-like structures containing distinct PAS-positive granules were first seen after 3 days of infection with tachyzoites, and became numerous by 6 days. Argyrophilic walls were first seen after 6 days, and became numerous by 16 days of infection with tachyzoites. Prepatent periods to oocyst shedding (PPO) were either “short” (3–10 days) or “long” (19–48 days). The “short” PPO was found only in cats that had ingested mice infected for 3 days or longer, and was related to the development of PAS-positive granules in T. gondii, and to high, 60–100%, oral infectivity rates for cats. The “long” PPO followed the ingestion of mice infected for only 1–2 days, and was related to tachyzoites without distinct PAS-positive granules and low, 32% or less, infectivity for cats. The “long” PPO followed also the ingestion of oocysts and the parenteral inoculation of tachyzoites, bradyzoites, or sporozoites. Using the “short” PPO as a criterion for detecting cysts in tissues, it was shown that (a) numerous cysts developed in mice 5 days after inoculation with tachyzoites, 7–9 days after inoculation with cysts, and 9–10 days after inoculation with oocysts, and (b) cysts developed faster and more frequently in the brain and muscle than in lungs, liver, spleen, and kidneys of mice inoculated with tachyzoites.     

Besnoitia darlingi (Apicomplexa,Sarcocystidae, Toxoplasmatinae): Transmission between Opossums and Cats1

ABSTRACT. Opossums (Didelphis marsupialis), act as intermediate hosts for Besnoitia darlingi and could be infected orally with sporozoites (oocysts) and bradyzoites (tissue cysts), or intraperitoneally (i.p.) with tachyzoites. Infections could presumably be transmitted through cannibalism. Cats (Felis catus), the definitive host, could be infected only with bradyzoites but not sporozoites. Oocysts shed by cats measure about 12 × 12 μm, resemble similarly sized oocysts of Toxoplasma gondii and Hammondia hammondi, and must be differentiated by the appearance of tissue cysts after experimental infection of intermediate hosts. Cats did not form tissue cysts of B. darlingi. Tachyzoites from the related B. jellisoni could be used in the Sabin-Feldman dye test to determine the development of antibody to B. darlingi in opossums after infection.     

Autofluorescence of Toxoplasma gondii and Neospora caninum cysts in vitro

Autofluorescence of Toxoplasma gondii and Neospora caninum was studied by fluorescence microscopy during their differentiation from tachyzoites to bradyzoites in vitro using Vero as host cells. Stage conversion into bradyzoites and cysts was confirmed by immunofluorescent microscopy and Western blot analysis using SAG1- and BAG1-specific antibody, respectively. From day 4 postinfection (PI), pale blue autofluorescence of the bradyzoites and tissue cysts was observed with UV light at 330-385 nm, which coincided with the onset of cyst development. This autofluorescence under UV light of bradyzoites and tissue cysts increased in intensity from days 8 to 10 PI. In contrast to the autofluorescence shown by bradyzoites and cysts, tachyzoites and parasitophorous vacuoles containing tachyzoites never autofluoresced at any time examined. Autofluorescence of the cystic stages was of sufficient intensity and duration to allow the detection of cysts and bradyzoites of T. gondii and N. caninum. In this study, we describe for the first time the autofluorescence properties of in vitro-induced bradyzoites and cysts of T. gondii and N. caninum.     

Ultrastructure of two types of first-generation merozoites of Eimeria bovis

Eimeria bovis has two types of first-generation merozoites with distinct ultrastructural characteristics. Type I merozoites were relatively large (means = 13.2 x 1.5 micron) and crescent-shaped, contained numerous micronemes and amylopectin granules, had a posteriorly located nucleus and a conical-shaped posterior tip, and were highly motile and capable of penetrating cultured cells. Type II merozoites were small (means = 5.9 x 0.9 micron) and spindle-shaped, had a centrally-located nucleus, few micronemes, few or no amylopectin granules, a dome-shaped posterior tip and little motility, and appeared to be incapable of penetrating cultured cells. It is possible that these two types of merozoites have considerably different roles in the life cycle of E. bovis.     

Immunohistochemical and ultrastructural evidence for Neospora caninum tissue cysts in skeletal muscles of naturally infected dogs and cattle.

Protozoan stages were detected in the skeletal muscles of four dogs suffering from neosporosis and two neonatal calves with confirmed Neospora caninum- infection which could be immunohistochemically labelled by an antiserum against the bradyzoite-specific antigen BAG-5. In one calf, a tissue cyst was labelled by an antiserum against the N. caninum isolate NC-1. Ultrastructurally, a 0.3-1 microm-thick cyst wall surrounded the labelled parasites. The cysts were located within myofibres and contained varying numbers of bradyzoites each measuring 5.2(+/-0.6) x 1.6(+/-0.3) microm. The encysted stages showed typical ultrastructural features of N. caninum bradyzoites with subterminal nuclei, electron-dense rhoptries, and micronemes that were orientated perpendicular to the zoite pellicle. Immunohistochemistry and serology did not reveal any evidence for co-infection with Toxoplasma gondii. The detection of tissue cysts in skeletal muscle of N. caninum-infected intermediate hosts is of major epidemiological importance for the understanding of how definitive or intermediate carnivorous hosts become infected with N. caninum.     

Ultrastructure of Two Types of First-Generation Merozoites of Eimeria bovis1

Eimeria bovis has two types of first-generation merozoites with distinct ultrastructural characteristics. Type I merozoites were relatively large (X = 13.2 times 1.5 μm) and crescent-shaped, contained numerous micronemes and amylopectin granules, had a posteriorly located nucleus and a conical-shaped posterior tip, and were highly motile and capable of penetrating cultured cells. Type II merozoites were small (X = 5.9 times 0.9 μm) and spindle-shaped, had a centrally-located nucleus, few micronemes, few or no amylopectin granules, a dome-shaped posterior tip and little motility, and appeared to be incapable of penetrating cultured cells. It is possible that these two types of merozoites have considerably different roles in the life cycle of E. bovis.     

Ultrastructure of the cyst of Sarcocystis muris.

Cysts of Sarcocystis muris develop within muscle cells and each is bounded by a parasitophorous vacuole membrane. Closely spaced spherical blebs formed from this membrane extend into the muscle cell cytoplasm. A dense substance fills the cavity of the bleb and occupies the vacuolar space immediately adjacent to the membrane. The remainder of the vacuole is filled with a moderately dense matrix within which the parasites develop. At 40 days after infection only metrocytes are present, characterized by their ovoid shape, lightly stained cytoplasm, amylopectin-like granules, and lack of micronemes. Metrocytes divide by a process resembling endodyogeny and eventually produce bradyzoites. By 78 days after infection, at which time the cyst is infective for cats, the few remaining metrocytes are located at the cyst periphery but most organisms are elongated and contain organalles characteristic for bradyzoites including micronemes, dense granules, and amylopectin. Structures indicative of division were not seen in bradyzoites. Rhoptries are few in number. Numerous vesicles of smooth endoplasmic reticulum accumulate in the cytoplasm of muscle cells adjacent to the periphery of the enlarging cyst but significant destruction of muscle fibers containing cysts with viable organisms was not seen in specimens fixed between 40 and 325 days after infection. Unusual lamellar structures were seen in some parasitized muscle cells and intracystic tubules occurred in some cysts.     

Neospora caninum: identification of 19-, 38-, and 40-kDa surface antigens and a 33-kDa dense granule antigen using monoclonal antibodies.

Neospora caninum, a coccidian parasite closely related to Toxoplasma gondii, can infect a broad host range and is regarded as an important cause of bovine abortion worldwide. In the present study, four antigens of N. caninum were partially characterized using monoclonal antibodies. Immunofluorescence of viable tachyzoites as well as the immunoprecipitation of antigens extracted from tachyzoites previously labeled by surface biotinylation revealed that three of these antigens with apparent molecular weights of 40, 38, and 19 kDa are located in the outer surface membrane of this parasite stage. Further evidence for the surface localization of the 38-kDa antigen was obtained by immunoelectron microscopy. In addition to the surface molecules, an antigen located in dense granules and in the tubular network of the parasitophorous vacuole was detected by another monoclonal antibody. When tachyzoite antigens separated under nonreducing conditions were probed on Western blots, this antibody reacted mainly with a 33-kDa antigen. Immunohistochemical analysis of infected tissue sections indicated that the 33-kDa dense granule antigen is present in both tachyzoites and bradyzoites, while the 38-kDa surface antigen from tachyzoites seems to be absent in bradyzoites.     

Ultrastructure of In Vivo-Produced Caryocysts Containing the Coccidian Caryospora bigenetica (Apicomplexa: Eimerüdae)

A cotton rat was inoculated orally with oocysts of Caryospora bigenetica from the feces of a rattlesnake. Sixteen days later the rat was euthanized, and portions of the scrotum, foot pad and muzzle were processed for histological sections and transmission electron microscopy. Sporozoites within caryocysts had typical coccidian features such as an anterior and posterior refractile body, centrally located nucleus, micronemes, rhoptries, a conoid, a micropore near the anterior refractile body, a posterior pore, amylopectin granules, lipid bodies, a Golgi-like body, a mitochondrion and subpellicular microtubules. The infected host cell was spherical and surrounded by a fibrous wall-like covering, 0.35–1.00 μm thick. This outer covering, when viewed in stained histological sections, was periodic acid-Schiff (PAS)-positive.     

Spontaneous cystogenesis in vitro of a Brazilian strain of Toxoplasma gondii

Conversion of Toxoplasma gondii tachyzoites to the bradyzoite stage and tissue cyst formation in the life cycle of the parasite have crucial roles in the establishment of chronic toxoplasmosis. In this work we investigated the in vitro cystogenesis and behavior of the EGS strain, isolated from human amniotic fluid. We observed that tachyzoites of the EGS strain converted to intracellular cysts spontaneously in LLC-MK₂ epithelial cells, HSFS fibroblasts and C6 glial cell lineage. The peak of conversion occurred in the LLC-MK₂ cells after 4 days of infection, when 72.3 ± 15.9 of the infected cells contained DBA positive cysts. Using specific markers against bradyzoite, tachyzoite and cyst wall components, we confirmed stage conversion and distinguished immature from mature cysts. It was also observed that the deposition of cyst wall components occurred before the total conversion of parasites. Transmission electron microscopy confirmed the fully conversion of parasites presenting the typical characteristics of bradyzoites as the posterior position of the nucleus and the presence of amylopectin granules. A thick cyst wall was also detected. Besides, the scanning microscopy revealed that the intracyst matrix tubules were shorter than those from the parasitophorous vacuole intravacuolar network and were immersed in a granular electron dense material. The EGS strain spontaneously forms high burden of cysts in cell culture without artificial stress conditions, and constitutes a useful tool to study this stage of the T. gondii life cycle.     

An alternative technique to reveal polysaccharides in Toxoplasma gondii tissue cysts

Ultrathin sections of tissue cysts isolated from the brain of Toxoplasma gondii infected mice were submitted to two different methodologies derived from the periodic acid - Schiff's reagent (PAS) technique. The use of osmium tetroxide vapor as a developing agent of the aldehyde oxidation to reveal polysaccharides with periodic acid resulted in positive reaction in amylopectin granules in bradyzoites, as well as in the wall and matrix of the cysts, with excellent increment of the ultrastructural morphology. This technique can be used for study of T. gondii-host cell intracellular cycle, the differentiation tachyzoite-bradyzoite, and also for the formation of cysts into the host cells.     

Is stage conversion the initiating event for reactivation of Toxoplasma gondii in brain tissue of AIDS patients?

Reactivation of chronic toxoplasmosis resulting in Toxoplasma encephalitis (TE) is a common event in acquired immune deficiency syndrome (AIDS) patients. Conversion from Toxoplasma gondii bradyzoites to tachyzoites is a prerequisite for reactivation. Until recently, the study of stage conversion in human tissue was not possible due to the lack of antibodies that recognize stage-specific epitopes after long-term formaldehyde fixation. Using the combination of a polyclonal anti-T. gondii antibody, the cyst-stage-specific monoclonal antibody CC2, and a tachyzoite-specific polyclonal antibody (anti-SAG1, recombinant), we tried to demonstrate parasite differentiation in the brain tissue of 10 AIDS patients with clinically suspected TE. Double labeling of the stage-specific antibodies enabled us to demonstrate interconversion between tachyzoites and bradyzoites for the first time in human tissue. The study confirmed that the transformation process is nonsynchronous and that the manifestation of TE depends on the degree and site of tissue destruction caused by invading tachyzoites. The original source of tachyzoites could never be located, but a few samples suggested that tachyzoites may invade by dissemination across the blood-brain barrier. Cyst rupture as the first event in the process of reactivation was not seen. We conclude that the initial site(s) of reactivation will be destroyed by tissue-destructive tachyzoites long before clinical symptoms occur.