Histochemistry and ultrastructure of the metacercarial cysts of blackspot trematodes Uvulifer ambloplitis and Neascus pyriformis

Cysts of Uvulifer ambloplitis from green sunfish, Lepomis cyanellus, and Neascus pyriformis from red shiners, Notropis lutrensis, were studied with light-level histochemistry and scanning and transmission electron microscopy. Cysts of both species are bilayered, consisting of an outer host capsule and an inner parasite cyst; the space between these layers is filled with a viscous material. The outer portion of the host capsule of both species is composed of fibrocytes, melanin granules, and collagen fibrils, and the inner portion of layers of flattened fibrocytes. The parasite cyst of U. ambloplitis is formed of 2 layers, an outer dense layer and an inner light layer, whereas the parasite cyst of N. pyriformis is made of 3 layers. A thin outer light-staining layer is present in addition to the 2 layers observed in U. ambloplitis. Results of histochemical staining were the same for both species. The host capsule stained positively for proteins and neutral and acid mucopolysaccharides. The viscous material was positive for neutral and acid mucopolysaccharides but not for proteins. The parasite cyst gave a strong positive reaction for neutral mucopolysaccharides but was negative for acid mucopolysaccharides and proteins.     

Histochemical and ultrastructural studies on the metacercarial cyst of Echinostoma revolutum (Trematoda).

Histochemical and ultrastructural studies were made on the metacercarial cyst of Echinostoma revolutum obtained from the kidney of experimentally infected Physa and Lymnaea snails. Ultrastructural studies revealed three cyst walls, an outer, middle and inner. The outer wall was more electron-dense than the middle, and contained coarser granules than those found in the middle layer. The inner wall was lamellated and contained membranous whorls. Collagenous fibers presumably of host origin surrounded the outer cyst wall. The outer and middle cyst walls stained identically with all histochemical procedures used. These walls contained acid mucopolysaccharides and glycoprotein, whereas the inner cyst wall contained glycoprotein. All cyst walls stained positively with a variety of protein stains.     

The pathogenicity, localization, and cyst structure of echinostomatid metacercariae (Trematoda) infecting the kidneys of the frogs Rana clamitans and Rana pipiens

Light and scanning electron microscopy were used to examine the localization and pathogenicity of echinostomatid metacercariae infecting the kidneys of leopard frogs, Rana pipiens, and green frogs, Rana clamitans. Cysts occurred predominantly in the ventrolateral renal cortex, and at least some were confined to the lumen of the Bowman's capsules. Each vermiform metacercarial body was enclosed by a spherical cyst wall that had a uniform thickness. The wall was composed of a homogeneous material containing basic and keratinlike proteins, with sulfated acid mucopolysaccharides on the outer surface. Most cysts were enclosed by a fibrous capsule of host origin, or were surrounded by an inflammatory focus. Fibrosis was always focal, but its degree varied between individual hosts and between different cysts within the same host. Some heavily encapsulated cysts were darkened and contained disintegrating worms. In heavily infected kidneys, confluence of fibrotic or inflammatory foci resulted in the displacement of functional renal tissue. These data suggest that infection by echinostomatids may impair renal function and that the host's response affects parasite viability.     

The structure and composition of the cyst wall of the metacercaria of Cloacitrema narrabeenensis (Howell & Bearup, 1967) (Digenea: Philophthalmidae).

The mstacercarial cyst of Cloacitrema narrabeenensis which is formed in the open is composed of four layers: an outermost layer of acid mucopolysaccharide, a layer of protein which is presumed to be tanned, a layer of neutral mucopolysaccharide and an innermost layer of keratinized protein. The two layers which together form the outer cyst wall can be split off by slight pressure from the two remaining layers which together form the inner cyst wall. In the centre of the ventral side of the inner cyst wall, the keratinized layer is incomplete and this ventral plug region is composed of neutral mucopolysaccharide. The cyst wall is therefore very similar to that of Fasciola hepatica, the main difference being that the order of the two layers of the outer cyst is reversed. General evolutionary and functional relationships of metacercarial cysts are discussed.     

Structure and composition of the metacercarial cyst wall of Sphaeridiotrema globulus (Trematoda)

1986. Structure and composition of the metacercarial cyst wall of Sphaeridiotrema globulus (Trematoda). International Journal for Parasitology 16: 647–653. Histochemical and structural observations were made on the metacercarial cyst wall of Sphaeridiotrema globulus, obtained from naturally infected Goniobasis virginica (Pleuroceridae) snails. The cyst wall of S. globulus is a thin, glistening, transparent structure that thickens as the metacercaria ages. The cysts occur singly beneath the shell of the snail host or linked together in sheets, pyramids and other three dimensional forms. Light and electron microscopic examination of the cyst wall reveals an inner, middle and outer layer, each of which may vary in thickness. Adjacent cysts are linked by their outer layers. The chemical composition of these layers, elucidated through histochemistry, is described.     

The histochemistry of Stellantchasmus falcatus Onji and Nishio, 1915 (Trematoda: Heterophyidae) metacercarial cyst in the mullet Mugil cephalus L. and histopathological alterations in the host†

The metacercarial cyst of the heterophyid trematode Stellantchasmus falcatus in the striated muscles of the mullet Mugil cephalus consists of three layers. Histochemical studies have indicated that these layers are chemically distinct. The acellular inner layer is comprised of complex carbohydrate polyanions rich in acidic groups while the medial cellular layer is comprised of a neutral mucopolysaccharide including a moeity of carbohydrate poiyanions. These two layers are believed to be of parasite origin. The outer layer is cellular and is of host origin. It is the most chemically complex and is believed to include one or more of the following: mucopolysaccharides, glycolipids and phospholipids. In addition, the carbohydrate moeity associated with the mucopolysaccharides and/or glycolipids is in the form of sulphated carbohydrate polyanions with undissociated carboxyls as well as acidic groups. The presence of the parasite results in the necrotic degeneration of the host's myofibers situated in the proximity of the parasite. Large fat cells fill the spaces vacated by necrotic muscle cells and many blood cells occur along the periphery of the parasite.     

In vitro excystment of the metacercaria of Maritrema arenaria (Digenea: Microphallidae)

Metacercarial cysts of Mantrema arenaria were subjected to a solution containing trypsin and bile salts at 41°C. This treatment induced intense metacercarial activity and after 15 min metacercariae burst through their cyst walls and emerged. Electron microscopy demonstrated that during the process of excystment the inner layer of the cyst wall changed from a compact to a loose fibrous state. Experiments showed that only cysts containing viable metacercariae underwent this change whereas cysts which had been forcibly vacated before treatment did not. This indicated that the structural change of the inner layer of the cyst wall could not be attributed to the excystment medium. Also there was much less acid phosphatase activity in and on the surface of newly excysted metacercariae compared with encapsulated specimens. It was concluded that the excystment medium induced physical activity in, and the release of enzymic material by, the metacercariae. Together these activities rendered the cyst wall soft and susceptible to rupture by physical pressure.     

Stereoscan studies of rediae, cercariae, cysts, excysted metacercariae and migratory stages of Fasciola hepatica

The external surface of the redial body of Fasciola hepatica is provided with microvillus-like projections or short lamellae, and short cilium-like structures are common anteriorly. The anterior part of the cercarial body possesses a pattern of regularly arranged small depressions each containing a spine. Both long and short cilium-like structures occur anteriorly. The tail is spineless and provided with dorsolateral folds. The outer cyst wall is formed by granules secreted from the tegument all over the body apart from the ventral sucker. Most granules transform into fibrillae which form the thick outer spongy layer. The precursor of the inner cyst wall is at the beginning closely attached to the metacercarial surface, but later the membrane-like cyst wall extends, and when fully formed the metacercaria lies free in the flattened circular inner cyst. The ventral plug is formed by the ventral sucker. The tegument of newly excysted metacercariae is provided with simple pointed spines, but later during migration in the mouse the spines become flattened and multipointed. Very young migratory stages may be attached with host cells.     

Ultrastructure and histochemistry of the metacercarial cyst of Spelotrema nicolli (Microphallidae: Trematoda)

Based on electron microscopy and histochemical tests, four layers are described for the metacercarial cyst of Spelotrema nicolli. The outermost layer I is made up of host connective tissue. Layer II, which may contain lipid, is a narrow band varying in electron density. Layer III is composed of mucoprotein with a granular fine structure. The innermost layer is made up of columnar, proteinaceous crystals, which radiate out from the inner edge of the cyst. Observations on the epidermis of Spelotrema nicolli contrast previous work on the mechanism of cyst formation in microphallids.     

Metacercarial cyst formation in vitro of Echinostoma paraensei

Cercariae of Echinostoma paraensei Lie and Basch 1968 encysted normally in the presence of Biomphalaria glabrata embryo (Bge) cells in culture, partially in culture conditioned medium, and not at all in fresh culture medium alone. At the ultrastructural level the cyst is composed of 2 well defined regions. The outer cyst wall (OCW) is particulate to fibrous in nature, formed from secretory granules released from the cercarial tegument. Membranous scrolls or rodlets secreted from the subtegumental cystogenous gland cells are then added to this layer, forming the inner cyst wall (ICW). After 24 hr the cultured cyst is enclosed by a thin cellular capsule similar to that found around cysts in the snail host. The capsule also contains collagen fibers, not found elsewhere in Bge cell cultures.     

Histopathology, ultrastructure and immunohistochemistry of Coregonus lavaretus hearts naturally infected with Ichthyocotylurus erraticus (Trematoda)

Histopathological, ultrastructural and immunohistochemical investigations were conducted on 26 specimens of powan Coregonus lavaretus (L.) from Loch Lomond (Scotland). The hearts of all 26 powan (15 females and 11 males) investigated harboured metacercariae of the digenean trematode Ichthyocotylurus erraticus (Rudolphi, 1809). The vast majority of metacercariae were located either singly or as an aggregation of white cysts on the surface of the bulbus arteriosus. The intensity of infection ranged from 2 to 200 larvae heart(-1), although the number of metacercariae found on male powan did not exceed 13. Histochemically, the parasite cyst wall gave a strong positive reaction with periodic acid schiff (PAS) and a faint positive signal with Azan-Mallory stain. All the metacercariae cysts were embedded in a granulomatous proliferation of heart epicardium tissue, forming a reactive fibroconnective capsule around the parasite. The capsule enclosing the parasite (produced by the host's reaction to the parasite) measured 13.57 to 90.20 microm (37.43 +/- 3.56) in thickness. Within the capsule wall, eosinophilic granular cells (EGCs), granulocytes, melanocytes and, in some instances, partially degenerated or vacuolated epithelioid cells were observed in close proximity to the cyst wall. Pigment-bearing macrophages were scattered throughout the granulomatous host-tissue reaction and as macrophage aggregates (MAs) within the capsules surrounding parasites. Immunohistochemical tests were applied to infected heart sections using 12 different antisera. Nerve fibres immunoreactive to bombesin, substance P (SP), and atrial natriuretic peptide (ANP) antisera were observed in close proximity to the parasite larvae. The presence of a serotonin-like substance was also observed within host immune-cells surrounding trematode cysts. Large cells of the epicardium were found to be immunoreactive to met-enkephalin and vasoactive intestinal peptide (VIP) antisera but not immunoreactive to anti-protein gene-product 9.5 (PGP9.5) sera.     

Chemical, histochemical, and histopathological studies on corals. Porites spp., parasitized by trematode metacercariae

The occurrence of the metacercaria of Plagioporus sp. encysted in two species of corals, Porites compressa and P. lobata, from Kaneohe Bay, Oahu, Hawaii, is reported. This parasite is covered with a bilayered metacercarial cyst the chemical compositions of which have been ascertained by histochemistry. Both of these cyst walls are believed to be secreted by the parasite. In addition, the cytochemical properties of the metacercaria and the adjacent host cells are reported.Histo- and cytopathological alterations in the epidermis and gastrodermis of the coral hosts were studied and such changes have been interpreted to have resulted from mechanical pressure exerted by the parasite.Because gross observations had revealed the presence of elevated nodules at the sites where the metacercariae occurred, chemical analysis was conducted to ascertain whether this larval trematode had induced hypercalcification in the hosts. However, calcium determinations of Decal in which pieces of parasitized coral tissue had been decalcified revealed that hypercalcification had not occurred.Examination of the cellular reactions in both species of Porites revealed that if the gastrodermis had been ruptured, partial encapsulation of the parasite occurs. The reaction cells include free cells of the mesoglea that become fused, accidentally trapped cnidoblasts and nutritive-muscular cells, and gastrodermal interstitial cells. Despite the partial encapsulation, the encysted metacercariae did not appear to be injured.     

Ichthyophthirius multifiliis (Ciliophora) Invasion of Gill Epithelium1

The sequence of morphologic events associated with Ichthyophthirius rnultifliis invasion of gill epithelium began in the theront with differentiation of secretory mucocysts and the perforatorium. After escaping from the cyst the theront, which stained intensely with Mallory' stain, sought a host. As it approached the host epithelium, the contents of the mucocysts were discharged, enveloping the ciliate in sticky material, which made initial contact with the host epithelium. Rapid penetration by the theront caused disruption of one or more host cells and resulted in a focal necrosis associated with the anterior margin of the ciliate. Within five minutes postexposure, the parasite completed its invasion of the epithelial layer and stained less intensely. The remnants of host cells disrupted during its entry surrounded the trophont until they were ingested by the parasite. Within 40 min postexposure, synthetic activity of the parasite appeared to increase as evidenced by an abundance of organelles, particularly ribosomes and crystalline mucocysts. At this point, the overlying host epithelium appeared normal.     

Freeze-fracture studies of Cryptosporidium muris.

The attachment site of Cryptosporidium muris to host cells was investigated using the freeze-fracture method. Cryptosporidium muris was enveloped by a double membrane of host plasma membrane origin, which formed the parasitophorous vacuole. The outer membrane of the double membrane was continuous with the host plasma membrane at the dense band, while the inner membrane was connected with the anterior part of the parasite plasma membrane at the annular ring. The density of intramembranous particles (IMP) was dramatically altered at the above two junctures. The outer parasitophorous membrane showed low IMP-density as compared to the host plasma membrane, although both membranes were continuous. The inner parasitophorous membrane had few IMP, whereas the parasite plasma membrane showed numerous IMP. When the attachment sites of parasites and host cells were fractured, circular-shaped fractured faces were observed on both sites of the parasite and host cell. These exposed faces corresponded to the dense bands and were very similar in size in each parasite.     

Electron microscopic and cytochemical studies of the mouse subcommissural organ

Summary In mice most of the ependymal cells of the subcommissural organ (SCO cells) are densely packed with dilated cisternae of the endoplasmic reticulum (ER) containing either finely granular or flocculent materials. The well developed supra-nuclear Golgi apparatus consists of stacks of flattened saccules and small vesicles; the two or three outer Golgi saccules are moderately dilated and exhibit numerous fenestrations; occasional profiles suggesting the budding of coated vesicles and formation of membrane-bound dense bodies from the ends of the innermost Golgi saccules are seen. A few coated vesicles and membrane-bound dense bodies of various sizes and shapes are also found in the Golgi region.The contents of the dilated ER cisternae are stained with periodic acid-silver methenamine techniques. In the Golgi complex the two or three inner saccules are stained as deeply as the dense bodies, and the outer saccules are only slightly stained. The stained contents of ER cisternae are more electron opaque than those of the outer but less opaque than those of the inner Golgi saccules and the dense bodies.Acid phosphatase activities are localized in the dense bodies, some of the coated vesicles in the Golgi region, and in the one or two inner Golgi saccules.On the basis of these results the following conclusions have been reached: (1) In mouse SCO cells the finely granular and the flocculent materials in the lumen of ER cisternae contain a complex carbohydrate(s) which is secreted into the ventricle to form Reissner's fiber; (2) the secretory substance is assumed to be synthesized by the ER and stored in its cisternae, and the Golgi apparatus might play only a minor role, if any, in the elaboration of the secretory material; (3) most of the dense bodies in the mouse SCO cells are lysosomal in nature instead of being so-called dark secretory granules.Sponsored by the National Science Council, Republic of China.     

Histological reactions of the host induced by the larvae of the warble-fly Oestromyia leporina Pall. (Diptera, Hypodermatidae) (author's transl)

The histological reactions of Mastomys natalensis induced by the larvae of Oestromyia leporina are described from the start of the infection to the complete healing of the evacuated cyst. Against the migrating larva no cellular reactions take place. After the larva settles, the most obvious feature is a non-suppurative inflammation of the surrounding tissue, while a layer of granulation tissue, infiltrated with eosinophils, is built up around the parasite. About 13 days p.i. the number of eosinophils starts to decrease again. No giant cells are present; plasmocytes and lymphocytes are relatively scarce. A few days before the parasite leaves the host, the inner surface of the warble is infiltrated with masses of neutrophils, obviously caused by secondary invasion of bacteria through the warble opening. After the parasite leaves the host, the repair of the tissue takes place within three weeks. The remaining scar tissue contains cells carrying haemosiderin, which disappears entirely about two months later.     

Light and transmission electron microscopical studies and amino acid analysis of the metacercarial cyst of Zygocotyle lunata (Trematoda).

Light microscopical studies indicated that the cyst of Zygocotyle lunata consists of outer, inner and ventral cyst walls. Transmission electron microscopical studies showed that the outer cyst and the ventral cyst each consist of two layers. The inner cyst is lamellated and contains a specialized ventral region designated the ventral lid. Amino acid analysis of cyst walls showed only trace amounts of cysteine, indicating that disulphide bonds are not used to stabilize the inner cyst of Z. lunata.     

Ultrastructure of early stages of Rozella allomycis (Cryptomycota) infection of its host, Allomyces macrogynus (Blastocladiomycota)

This study reconstructs early stages of Rozella allomycis endoparasitic infection of its host, Allomyces macrogynus. Young thalli of A. macrogynus were inoculated with suspensions of R. allomycis zoospores and allowed to develop for 120 h. Infected thalli at intervals were fixed for electron microscopy and observed. Zoospores were attracted to host thalli, encysted on their surfaces, and penetrated their walls with an infection tube. The parasite cyst discharged its protoplast through an infection tube, which invaginated the host plasma membrane. The host plasma membrane then surrounded the parasite protoplast and formed a compartment confining it inside host cytoplasm. The earliest host-parasite interface within host cytoplasm consisted of two membranes, the outer layer the host plasma membrane and the inner layer the parasite plasma membrane. At first a wide space separated the two membranes and no material was observed within this space. Later, as the endoparasite thallus expanded within the compartment, the two membranes became closely appressed. As the endoparasite thallus continued to enlarge, the interface developed into three membrane layers. Thus, host plasma membrane surrounded the parasite protoplast initially without the parasite having to pierce the host plasma membrane for entry. Significantly, host-derived membrane was at the interface throughout development.     

Acid-Fast Staining for Hooklets of Echinococcus

Recognition of well-developed hydatid disease in tissue sections or in aspirated cyst fluid usually presents no difficulties. The distinctively laminated outer capsule, the active inner germinative layer and the hydatid fluid containing characteristic brood capsules, scolices and free hooklets, are outstandingly reliable diagnostic criteria. The identification of the organism assumes more difficulty in old, arrested infections when the outer layer, and even the surrounding cellular inflammatory response, has been replaced by a wall of nonspecific, collagenous tissue and the cyst contents contain only calcified remnants of scolices. In the course of time, even the pathognomonic hooklets may no longer be recognizable.     

Acid-Fast Staining for Hooklets of Echinococcus

Recognition of well-developed hydatid disease in tissue sections or in aspirated cyst fluid usually presents no difficulties. The distinctively laminated outer capsule, the active inner germinative layer and the hydatid fluid containing characteristic brood capsules, scolices and free hooklets, are outstandingly reliable diagnostic criteria. The identification of the organism assumes more difficulty in old, arrested infections when the outer layer, and even the surrounding cellular inflammatory response, has been replaced by a wall of nonspecific, collagenous tissue and the cyst contents contain only calcified remnants of scolices. In the course of time, even the pathognomonic hooklets may no longer be recognizable.