Light microscope and TEM structure of tissue-cysts in the connective tissues of the lizard

Fresh and stained preparations of the cyst contents revealed vast numbers of bradyzoites (Fig. 1). In histological sections, the cyst wall is seen to have been reduced to a narrow homogenous wall enclosing the closely packed bradyzoites.

Fig. 1. Besnoitia sp., in the teiid lizard Ameiva ameiva. Bradyzoites in a smear made from a ruptured tissue cyst. Bouin fixation/Giemsa staining.

By TEM the wall was shown to be comprised of an inner cytoplasmic (‘host cell’) layer and an external fibrous layer (‘cyst wall’ of Sheffield, 1968). The densely granulated cytoplasmic layer contained either a single multi-lobed nucleus, or several grossly hypertrophic nuclei (Fig. 2A). Its contents varied in thickness and density from cyst to cyst and contained variable proportions of rough and smooth endoplasmic reticulum (ER), cisternae, lipid vacuoles, and mitochondria (Fig. 2A and B): in some cysts, the rough ER aggregates into concentric formations (Fig. 2C). The plasma membrane on the PV boundary is protruded into very delicate tubule-like extensions which form a distinct layer on the surface of the PV wall. A few cytoplasmic protrusions penetrate, sometimes deeply, into the apposed external fibrous layer (Fig. 2A). This outer layer contained longitudinal and transverse filamentous strands within variable amounts of ground substance: in older cysts these microfibrils may be in disarray (Fig. 2C). The fibrous material in some cyst walls appears to be organized into 2 layers (Fig. 2A).

Fig. 2. (A–E) Fine structure of a tissue cyst from Ameiva ameiva. (A) Overall view of the cyst wall layers: the cytoplasmic (‘host-cell’) (C), its nuclei (N) and the PV-lining tubuli (T); extension of the cytoplasmic layer into the ‘cyst wall’ (pr) and ‘cyst wall’ layers (W). A gap in the cyst wall is marked with an arrow. (B) Details of the cytoplasmic (C) and the PV-lining tubuli layers (T): rough (rer) and smooth (er) endoplasmic reticulum, mitochondria (t), lipid vacuoles (L) and vesicles (v). (C) RER (arrow) concentrically arranged in the cytoplasmic layer (C), the cyst wall (W) seems to undergo shrinking (aging). (D) Dividing parasite with a primordium of daughter cell (arrow), a, apical complex, r. rhoptry. (E) Bradyzoite with elongate rhoptries (r) and ‘enigmatic bodies (arrows).

Within the PV most of the parasites are fully developed bradyzoites, with a few dividing by endodyogeny and containing primordia of daughter cells. The latter had a distinct conoid, a few rhoptries (not readily seen in section planes), while micronemes were lacking (Fig. 2D). The bradyzoites possessed a distinct conoid, elongate rhoptries and numerous micronemes which fill the entire pre-nuclear zone: they also contained the ‘enigmatic bodies’ as described by Senaud (1969), electron-dense globules and amylopectin granules (Fig. 2E).

Infection in laboratory mice and hamsters

From 7–14 days after intraperitoneal inoculation of the cyst contents, mice showed signs of sickness, with ruffled fur, diminished activity and increasing ascites. Some mice died with very abundant ascitic fluid containing variable numbers of intracellular and free parasites (tachyzoites) (Fig. 3A and B). Surviving mice recovered their normal appearance and activity but, in all those examined, possessed developing cysts in the tissues of their viscera and mesenteries. Hamsters were shown to develop similar infections. Some died from the acute infection.

Fig. 3. (A and B) Paired and quadruple sets of tachyzoites in the peritoneal exudate of a laboratory mouse, 7 days after the intraperitoneal inoculation of bradyzoites, Bouin/Giemsa staining.

TEM of tachyzoites in the peritoneal fluid cells of mice, 8 days p.i.

Host cells were predominantly neutrophils and monocytes, which contained single organisms (Fig. 4A) in a state of endodyogeny (Fig. 4B), or the products of such division (Fig. 4C). All stages possessed a bilayered pellicle, and were lodged in a parasitophorous vacuole (PV) bounded by a single unit membrane which was sometimes fragmented (Fig. 4A and B). Some tachyzoites were seen to be coated by a glycocalyx-like aggregate of a granular nature. The PV contained variable amounts of tubular whorls (Fig. 4A) mixed with a globular residue (Fig. 4B): sometimes it also contained lysosomal fragments. In some of the host cells the mitochondria and Golgi elements were seen to be concentrated at the cytoplasmic periphery of the PV. Where the PV boundary was indiscernible the outlining host cell cytoplasm also appeared to be disintegrating, with the material appearing to become continuous with that of the PV (Fig. 4C).

Fig. 4. (A–D) TEM of tachyzoites in the peritoneal fluid cells (monocytes) of mice, 8 days p.i. (A) Single tachyzoite in a host cell (H), the PV wall is discontinuous (arrows), and sometimes aligned by ER(er); PV contains tubular whorls (tw). (B) Tachyzoite dividing by endodyogeny (ga, granular aggregates; Ht, host-cell mitochondrium; m, micronemes, pp, progeny primordia; r, rhoptries; t, parasite mitochondrium). (C) Progeny of endodyogeny (a, apical complex; ga, granular aggregates; r, rhoptries). (D) Progeny of polyendyogeny.

The conoid of the tachyzoites was accompanied by a circle of elongate rhoptries, which extended beyond mid-length of the entire organism (Fig. 4C). Micronemes were seen in the single and the dividing tachyzoites (Fig. 4A and B) but seemed to be very few in their offspring. A layer of electron-dense substance was accumulated in the most anterior part of the subpellicular zone (Fig. 4C). Eight microtubules could be traced in longitudinal sections of the tachyzoites. All of the organisms, and their progeny, contained either 1 large mitochondrion or several smaller ones. Those in various stages of endodyogeny contained either rudimentary offspring or more advanced ones with an apical complex (Fig. 4B). Rounded, granular aggregates (ribosomes?), which are present only in the daughter cells (Fig. 4C), could already be traced in the endodyogeny primordia (Fig. 4B). An accumulation of 8 tachyzoites seen in a single PV (Fig. 4D) suggests the additional existence of merogonous (polyendogenous) division. Products of this division contained many rhoptries and micronemes.

Stages of tissue cysts in mice

In a light microscopical study of sectioned tissues of mice sacrificed (or dying) up to 25 days p.i., developing tissue cysts found in the lungs, stomach serosa and mesenteries (Fig. 5A and B) measured up to 100 μm in diameter, while in other mice killed at 30–34 days p.i. some cysts had reached approximately 130×100 μm (Fig. 5C). The cyst wall seen at first to be composed of an inner cytoplasmic zone with numerous hypertrophic and segmented nuclei, each with several small nucleoli, and a peripheral non-nucleated and more hyaline layer. The 2 layers were separated by a space containing many minute, dark granules; the PV was filled with divided stages (Fig. 5A). With further development the PV, packed with parasite progeny, increased in volume and both the cyst wall inner nucleate and the apposing outer layers became compressed (Fig. 5B). Further increase in the cyst volume resulted in compression of the wall till the inner details of the 2 layers become only vaguely distinguishable (Fig. 5C). There were no notable changes in the host's surrounding tissues.

Fig. 5. (A–D) Histological view of stages of tissue-cysts in mice. (A) Young cyst (25 days p.i.) enclosing PV filled with products of endodyogeny, the nucleate cytoplasmic layer (C) clearly separated by a gap filled with fine granules (arrow) from the ‘cyst wall’ (W); (B) On-growing cyst (30 days p.i.), with the cytoplasmic, nucleate layer (C) being compressed against the ‘cyst wall’ (W). (C) Further differentiated cyst (30 days p.i.), with its compressed wall components. (D) TEM view of walls of tissue cysts in experimentally infected mice (30 days p.i.) (C, cytoplasmic layer; H, host connective tissue; pr, protrusions of the cytoplasmic layer; PV, parasitophorous vacuole, rer, rough ER; t, mitochondria; W, cyst wall).

TEM of tissue cysts from a mouse 30 days p.i.

The tissue cysts were at different stages of differentiation but demonstrated the same configuration of a PV filled with the products of endodyogeny enclosed by an inner cytoplasmic wall (‘host cell’) containing several large, compact or multilobed nuclei and an apposed outer, fibrous wall of 1 or more, layers (‘cyst wall’) (Figs 5D and 6A, B). The inner cytoplasmic layer contained rough ER, either concentrically tightly packed (Figs 5D and 6A), or loose and disarrayed (Fig. 6B). The spaces between the ER contained variable quantities of mitochondria (Figs 5D and 6C). ER elements were absent between the nuclei and the PV boundary, and in the septum extending to divide the PV of some cysts into 2 compartments. The plasma membrane on the PV boundary, similar to that seen in cysts in the lizard host, formed a layer of very delicate tubule-like extensions lining the PV wall surface (Fig. 6B). The membraneous boundary of the inner cytoplasmic layer was seen to send both short and elongated protrusions into the adjoining outer fibrous layer: these protrusions seemingly contained extensions of the ER (Figs 5D and 6A). The outer, fibrous layers of the cyst wall contained fibrillar bundles, sometimes orientated perpendicularly, and a narrow gap containing a fine ground substance separating the main fibrous layer from an outer narrower one (Fig. 5D).

Fig. 6. (A–C) TEM view of tissue cysts in experimentally infected mice (30 days p.i.), pr, protrusions of the cytoplasmic layer scale. (A) Enlarged view of the cytoplasmic wall with concentric rough ER, arrow head, PV-lining tubuli layer. (B) Cytoplasmic wall with mitochondria (t) and scattered rough ER (arrow heads). (C) Dividing parasite (T) and bradyocytes (B) in the PV of the tissue cyst shown in Fig. 5D; r, rhoptries.

The PV of each cyst contained bradyzoites, as well as a few stages undergoing division by endodyogeny (Fig. 6C). Dividing stages had rhoptries but lacked micronemes. Some bradyzoites were showing the expanded posterior end, with pellicular thickenings (‘posterior ring’ of Sheffield, 1968) characteristic of the bradyzoites' site of detachment from the mother cell.

Attempts to indicate the definitive host

No oocysts characteristic of Besnoitia were detected in faeces from the 3 kittens 4–15 days after they were fed with tissue cysts of chronically infected mice, or in fresh preparations of scrapings along the length of the intestine of each animal on autopsy. Similarly, no evidence of an induced infection could be found in the young boa-constrictor.