Raillietiniaisis in a Toddler From Hawaii: A Case of Mistaken Tapeworm Identity.

We present the first reported case from the United States of human infection with the intestinal cestode Raillietina, a parasite primarily of rodents and poultry. As the differential diagnosis between the morphologically similar Dipylidium caninum was challenging, we discuss the biology, epidemiology, and clinical management of this rare parasite.

Echinococcus multilocularis Leuckart, 1863 (Taeniidae): new data on sperm ultrastructure.

The present study establishes the ultrastructural organisation of the mature spermatozoon of Echinococcus multilocularis, which is essential for future research on the location of specific proteins involved in the sperm development in this species and also in Echinococcus granulosus. Thus, the ultrastructural characteristics of the sperm cell are described by means of transmission electron microscopy. The spermatozoon of E. multilocularis is a filiform cell, which is tapered at both extremities and lacks mitochondria. It exhibits all the characteristics of type VII spermatozoon of tapeworms, namely a single axoneme, crested bodies, spiralled cortical microtubules and nucleus, a periaxonemal sheath and intracytoplasmic walls. Other characteristics observed in the male gamete are the presence of a >900-nm long apical cone in its anterior extremity and only the axoneme in its posterior extremity. The ultrastructural characters of the spermatozoon of E. multilocularis are compared with those of other cestodes studied to date, with particular emphasis on representatives of the genus Taenia. The most interesting finding concerns the presence of two helical crested bodies in E. multilocularis while in the studied species of Taenia, there is only one crested body. Future ultrastructural studies of other species of the genus Echinococcus would be of particular interest in order to confirm whether or not the presence of two crested bodies is a characteristic of this genus.

Evidence of apoptosis in Raillietina echinobothrida induced by methanolic extracts of three traditional medicinal plants of Northeast India.

The therapeutic benefits of medicinal plants in terms of anthelmintic properties are known since time immemorial in India, particularly among natives of the Northeast India. However, only sporadic and scarce reports on scientific validation of these plants are available. The present study was conducted on the cestode Raillietina echinobothrida, to establish whether the anthelmintic activity of Potentilla fulgens, Alpinia nigra and Millettia pachycarpa was mediated by apoptosis or not. Light microscopic observation following MTT assay revealed the highest percentage of inhibition of viability among the worms by methanol extract of M. pachycarpa (89.33%), followed by A. nigra (65%) and P. fulgens (37%). Ultrastructural observations revealed swelling of mitochondria, disruption of mitochondrial membrane, vacuolization of mitochondria, appearance of apoptotic bodies in the cytoplasm, disintegration of nuclear membrane and nucleolus were very common throughout the tegument. DAPI stained specimens showed typical morphology of apoptosis, like nuclear condensation and fragmentation in the extracts treated parasites. A decrease in mitochondrial membrane potential was also recorded in the treated groups. Confirmatory TUNEL assay and DNA fragmentation assay of the extracts treated parasites also confirmed the apoptotic nature of cell death and is concluded to be responsible for paralysis and death of the parasite.

Cytocomposition of the vitellarium in Khawia sinensis Hsü, 1935 (Cestoda, Caryophyllidea, Lytocestidae): another caryophyllidean species with lamellar bodies and lipids.

The vitellarium of the invasive caryophyllidean tapeworm Khawia sinensis Hsü, 1935 from carp Cyprinus carpio L. was examined by means of transmission electron microscopy and cytochemical staining for glycogen with periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-SP). A vitellarium consists of numerous follicles of irregular size that are interconnected by a net of vitelline ducts. Vitelline follicles are composed of vitelline cells at various stages of development that are interconnected by interstitial tissue. Vitelline follicles are surrounded by a cytoplasmic sheath associated with an intercellular matrix. Extensive development of the granular endoplasmic reticulum and Golgi complexes are both involved in the production of shell globules/shell globule clusters and characterise cytodifferentiation of vitellocytes. Nuclear and nucleolar transformation lead to the formation and storage of intranuclear glycogen, a feature specific for the Caryophyllidea. Newly observed within the mature vitellocytes of Khawia sp. is the presence of lamellar bodies and a few lipid droplets. These cytoplasmic inclusions first occur in the mature cells within the follicles and persist in the vitelline cells within vitelloducts and intrauterine eggs. Two types of lamellar bodies are detected: regular lamellar-structured body and irregular lamellar-structured body. None of the lamellar bodies are membrane bound. Results of the present study indicate that the formation of lamellar bodies may be closely related to the endoplasmic reticulum or shell globule clusters. Some of the shell globule clusters are transformed into lamellar body clusters. Ultrastructural features of vitellocytes in K. sinensis are compared with those of other monopleuroid, polypleuroid, and strobilated cestodes.

Revisiting caryophyllidean type of spermiogenesis in the Eucestoda based on spermatozoon differentiation and ultrastructure of Caryophyllaeus laticeps (Pallas, 1781).

Spermiogenesis of the monozoic cestode Caryophyllaeus laticeps (Pallas, 1781) (Caryophyllidea: Caryophyllaeidae), a parasite of Abramis brama (Pisces: Cyprinidae), has been investigated by means of transmission electron microscopy and cytochemical staining with periodic acid-thiosemicarbazide-silver proteinate for glycogen. The process of spermatozoon formation corresponds in the basic pattern to that of Khawia armeniaca as described by Brunanská and Poddubnaya (Parasitol Res 99:449-454, 2006). The dense material at the early stages of spermiogenesis, an intercentriolar body, the formation of a free flagellum and flagellar bud, the penetration of the nucleus into the spermatid body when the fusion of the free flagellum with the median cytoplasmic process has started, and a complete proximodistal fusion have been determined. In contrast to previous data on the caryophyllidean type spermiogenesis, the latter more recent observations show the presence and the rotation of a free flagellum and a flagellar bud. This pattern indicates clearly a derived stage of spermiogenesis in the Caryophyllidea, when the second shorter flagellum is greatly reduced comparing with that in the Spathebothriidea, Diphyllobothriidea, or Bothriocephalidea, thus forming a flagellar bud. The flagellar bud occurs in all stages of spermiogenesis and represents an evolved character in the Caryophyllidea. The mature spermatozoon of C. laticeps consists of one axoneme of the 9 + "1" trepaxonematan structure, parallel cortical microtubules, and a nucleus. Cell components are situated in a moderately electrondense cytoplasm, containing glycogen in the principal regions (II, III, and IV) of the spermatozoon. A crested body is absent. Similarities and differences between spermatozoa of caryophyllideans as well as other Eucestoda are discussed.

Classical and molecular cytogenetics of Khawia sinensis (Cestoda: Caryophyllidea), invasive parasite of carp, Cyprinus carpio.

Chromosomes of the invasive tapeworm Khawia sinensis (Caryophyllidea), the specific parasite of common carp, were analyzed by means of conventional Giemsa staining and using fluorescent DAPI and YOYO-1 dyes, silver staining, and fluorescent in situ hybridization (FISH) with 18S rDNA probe. The karyotype is composed of eight pairs of metacentric and telocentric chromosomes (2n = 16, n = 3m + 5t, TCL = 42.54 µm). Constitutive heterochromatin was located at pericentromeric regions of all pairs, except for the largest metacentric pair (no. 1), which possessed no DAPI-positive band. FISH with rDNA probe revealed that both homologues of chromosome pair no. 6 carry a cluster of ribosomal arrays, which were located interstitially close to the centromere. Present results are compared with previous cytogenetic data on Khawia spp., and comments are made on the karyotypes with respect to their phylogenetic links.

Ultrastructure of the rhyncheal apparatus and other structures of the scolex of Grillotia (Christianella) carvajalregorum (Cestoda: Trypanorhyncha).

The scolex ultrastructure was studied in Grillotia (Christianella) carvajalregorum (Cestoda: Trypanorhyncha) using histochemistry and transmission electron microscopy. We show for the first time the presence of scolex glands arranged in two longitudinal acini at the pars vaginalis parenchyma. These glands, along with those scattered in bothrial parenchyma, produce potentially adhesive glycoprotein secretions that are discharged via ducts to the bothrial grooves and apex. A particular type of sensory receptor was found around frontal gland pores, with a possible function in regulating their secretion activity. The internal structure of microtriches varies according to their morphotype and distribution on the scolex, this study providing the first description of the ultrastructure of serrate lanceolate spinitriches. The projections that form serrate margins are an extension of the medulla, differing from similar projections of other spinitriches. The large caps observed in serrate lanceolate spinitriches may reflect their specialization in attachment to and abrasion of intestinal mucosa, while the short caps and large bases of acicular filitriches may reflect their involvement in nutrient absorption. We also describe the rhyncheal apparatus ultrastructure, showing a similar basic structure of tentacular walls than that of other trypanorhynchs. Some differences among species in the number of fibrous layers, composition of the apical cytoplasm and presence of microvilli-like projections were discussed. Finally, our study describes in detail the internal ultrastructure of hollow hooks, evidencing the presence of cytoplasm, mitochondria and fibrils. The location of these fibrils may increase the area of contact surface of hooks on tentacles, possibly allowing for a higher tensile strength than that of solid hooks. We consider that gland location and shape, composition of tentacular wall layers, and hook internal structure may serve as useful characters for the taxonomy and phylogeny of Trypanorhyncha. RESEARCH HIGHLIGHTS: This is the first description of scolex internal ultrastructure in Grillotia carvajalregorum, showing the presence of glands arranged in two longitudinal acini at the pars vaginalis parenchyma, with potentially adhesive functions. The internal ultrastructure of serrate lanceolate spinitriches and acicular filitriches may reflect their specialization in attachment to the host intestinal mucosa and their involvement in nutrient absorption, respectively. Internally, hollow hooks have cytoplasm with mitochondria and fibrils, which are more widely distributed than in solid hooks, possibly increasing their tensile strength.

Vitellogenesis in the cestode Atractolytocestus huronensis Anthony, 1958 (Caryophyllidea: Lytocestidae).

Vitellogenesis in the parthenogenetic caryophyllidean tapeworm Atractolytocestus huronensis Anthony, 1958, parasitizing the carp Cyprinus carpio L., has been examined using light (LM) and transmission electron microscopy and cytochemical staining with periodic acid-thiosemicarbazide-silver proteinate for glycogen. It was found that formation of vitellocytes shares the same basic patterns in lower tapeworms with parthenogenetic and normal reproduction. Each vitelline follicle of A. huronensis consists of vitellocytes at various stages of development and an interstitial tissue. Projections of the interstitial tissue surround each vitellocyte and extend as a cytoplasmic sheath on the follicular periphery. In contrary to other Eucestoda, interstitial tissue of A. huronensis includes numerous electron-dense vesicles of various sizes. Maturing and mature vitellocytes contain vitelline material in the form of single small shell globules, which may gradually fuse and give rise to the large shell globule clusters. Shell globule clusters are of two types in A. huronensis. Additional vitelline material is represented by single "lamellar" granules and glycogen in the cytoplasm of mature vitellocytes. The ultrastructural features of vitellogenesis and/or vitellocyte in A. huronensis are compared with that in other caryophyllideans and/or Neodermata.

New data on spermiogenesis and trepaxonematan axoneme in basal tapeworms (Cestoda, Caryophyllidea, Lytocestidae) parasitizing cyprinid fishes.

Monozoic caryophyllidean cestodes, intestinal parasites of cyprinid fishes, represent a group of tapeworms with an unclear evolutionary history. As spermatology may provide phylogenetically important data, the spermiogenesis and ultrastructure of the mature spermatozoon have been investigated using an integrative approach combining transmission electron microscopy, cytochemistry and electron tomography in Khawia rossittensis (Szidat, 1937). The process of spermatid formation is accompanied by the presence of ultrastructural characters not described in traditional models of spermiogenesis, e.g., apical electron-dense material, the two striated roots situated unusually opposite each other, branching of typical striated roots, an intercentriolar body comprising five electron-dense and four electron-lucent layers, rotation of both free flagella and flagellar buds to the median cytoplasmic process at 90°, and a complete proximodistal fusion. The synchronous rotation of both flagellar buds and growing free flagella is an evolutionarily linked pattern favouring the hypothesis that the Caryophyllidea are not ancestral but are secondarily derived from polyzoic forms. Electron tomography analysis has revealed a unique feature of two helicoidal tubular structures in the central electron-dense core of the axoneme of mature spermatozoon. These data provide new insights into the architecture of the 9 + '1' axoneme, which is shared by male gametes of all trepaxonematan Platyhelminthes.

Pixel by pixel: real-time observation and quantification of passive flotation speeds of three common equine endoparasite egg types.

The efficacy of anthelmintic treatments against populations of endoparasites infecting livestock throughout the world is decreasing. To mitigate this, the use of fecal egg counts is recommended to determine both the necessity, and to ensure the appropriate choice, of anthelmintic treatment. Traditionally, and in order to facilitate easier identification and/or enumeration, samples are analysed after separating eggs from other fecal particulates by exposing them to a solution with a density higher than that of the eggs, but lower than the remaining fecal contents. While many parasite egg flotation protocols exist, little is known about the characteristics of these eggs with respect to their movement through a flotation solution. In this study, we have demonstrated a novel method for the observation and quantification of microscopic (65-100 µm) objects as they experience unassisted flotation. This also represents, to our knowledge for the first time, that the flotation of parasite eggs has been observed and their movement characteristics quantified as they float through solution. Particle tracking and video analysis software were utilised to automatically detect and track the movement of individual eggs as they floated. Three 30 s videos and one 2 min video of each egg type were analysed. If the first 30 s of video were discounted, the differences in mean flotation speed among all videos was statistically significant between egg types (P = 0.0004). Strongyle type eggs (n = 201) moved the fastest with a mean 51.08 µm/s (95% confidence interval: 47.54-54.62). This was followed by Parascaris spp. (n = 131) and Anoplocephala perfoliata eggs (n = 322), with mean speeds of 44.43 µm/s (95% confidence interval: 39.47-49.4) and 31.11 µm/s (95% confidence interval: 29.6-32.61), respectively. This method for evaluating the mean speed of passive flotation may represent a first step towards further optimizing fecal egg flotation and be of interest to parasitologists and veterinary practitioners.

Do glial cells exist in the nervous system of parasitic and free-living flatworms? An ultrastructural and immunocytochemical investigation.

It is still unclear whether flatworms have specialized glial cells. At present there are no special methods available for the identification of glial cells in flatworms. The aim of this research was to carry out detailed investigations of the CNS in two species ofcestodes, and to get an idea whether these cells may fit into the concept of glia. Three types of glial cells have been found in Grillotia erinaceus: (1) fibroblast-like cells in the cerebral ganglion (CG); (2) glial cells in bulbar nerves with filaments and laminar cytoplasm; (3) a 3rd type of cells forms multilayer envelopes in the main cords (MC); also they make contacts with the excretory epithelium. To demonstrate the existence of glial cells, an immunocytochemical and ultrastructural investigation of Ligula intestinalis was undertaken. Intensive S100b-like immunoreaction (IR) was found in the GG and in the MC. IR-varicosities were mostly located asymmetrically on the MC, and no IR was found in neuropiles. Small glial cells were found on the surface of the MC; they have oval nuclei and dense cytoplasm with slim processes going around the neuropile and enclosing neurons. Long junctions are seen between cell processes but with neurons they usually possess juxtaposition contacts. Glial cells lack vesicles or synapse-like structures. Intensive S100b-like-IR has been shown in the CNS of cestodes for the first time. Results from ultrastructural research support the immunocytochemical date.

Cellular organization of the oncosphere of Mosgovoyia ctenoides (Cestoda: Anoplocephalidae).

The ultrastructure of the infective oncosphere of the cestode Mosgovoyia ctenoides (Anoplocephalidae) is described. The surface of the infective oncosphere is covered by a thin cytoplasmic layer of tegument connected by a narrow cytoplasmic process with the binucleate subtegumental cell, situated deeper in the body. Below the basal matrix of the cytoplasmic layer of the tegument are situated wide bands of the peripheral, somatic musculature responsible for body movements. The 3 pairs of hooks and their muscles form a complex hook muscle system, responsible for coordinated hook action. Five major types of cells have been distinguished: (1) a binucleate subtegumental cell, (2) a binucleate penetration gland, (3) 2 nerve cells, (4) numerous somatic cells, and (5) about 6 germinative cells. The approximate number of cells is 24 (26 nuclei, including 2 syncytial structures). The results of this study, when compared with other published reports from other cestode taxa, support previous hypotheses that the progressive reduction of oncosphere cells is an adaptive feature in cestode evolution.

Vitellogenesis of diphyllobothriidean cestodes (Platyhelminthes).

The recently erected cestode order Diphyllobothriidea is unique among all tapeworm orders in that its species infect all major groups of tetrapods, including man. In the present paper, the vitellogenesis of representatives of all three currently recognized families of this order was evaluated, based on ultrastructural (transmission electron microscopy) and cytochemical (detection of glycogen) observations. Vitelline follicles of all taxa studied, i.e. Cephalochlamys namaquensis from clawed frogs (Xenopus), Duthiersia expansa from monitors (Varanus) and Schistocephalus solidus that matures in fish-eating birds, contain vitelline cells at various stages of development and interstitial cells. Developing vitellocytes are characterized by the presence of mitochondria, granular endoplasmic reticulum and Golgi complexes involved in the synthesis of shell globules and formation of shell globule clusters. Mature vitellocytes contain lipids and glycogen in different proportions. The most significant differences among the three diphyllobothriidean families were found in the presence or absence of lamellar bodies. Variations of vitelline clusters morphology and types of lipid droplets are described and discussed in relation to the presumed evolutionary history of diphyllobothriideans, which belong to the most basal cestode groups.

Ultrastructure of the scolex and tentacles of the metacestode of Polypocephalus species (Cestoda: Lecanicephalidae) from the blue-swimmer crab Portunus pelagicus.

The ultrastructure and histochemistry of the scolex of a Polypocephalus species is described. This cestode is distinguishable from all known Polypocephalus spp. in that it has approximately 400 tentacles and inhabits the musculature of the blue swimmer crab, Portunus pelagicus. The metacestode has a large scolex, divided into an anterior par apicalis, and a posterior pars basalis. An elongate cercomer lies posteriorly to the scolex. The pars basalis lacks accessory suckers. The tegument of this region is armed with numerous spinous microtiches. The pars apicalis consists of approximately 400 unarmed tentacles, a large cavity ( = scolex cavity) containing some glandular tissue, and lying posteriorly to this cavity, a large gland ( = scolex gland). The tegument of the tentacles and pars apicalis is typically syncytial, and contains abundant electron-opaque secretory vesicles. These vesicles are produced by glandular tissue of the scolex gland and scolex cavity. Histochemistry revealed the presence of proteins and neutral mucosubstances in tentacles, scolex gland and scolex cavity, suggesting the presence of glycoproteins. It is argued that the secretion produced by the tentacles has an adhesive role. When applied to the large interface between the tentacles and the mucosa of the hosts, this secretion may provide the adult parasite with a strong holdfast to prevent dislodgement.

Immunolabelling of fish host molecules on the tegumental surface of Ligula intestinalis (Cestoda: Pseudophyllidea).

Immunoblotting, SDS-PAGE and western blotting procedures were used to demonstrate cross-reactivity of a polyclonal anti-carp IgM antibody with components of roach serum. The polyclonal antibody labelled 2 major bands in both immune and normal roach sera corresponding to molecular masses of approximately 90 and 65 kDa. One of these bands (65 kDa) was considered to be heavy chain of fish immunoglobulin whilst the identity of the other remains uncertain. This cross-reaction has been exploited in immunofluorescence and immunogold labelling studies to localize fish host molecules on the tegumental membrane of Ligula intestinalis freshly removed from roach fry. Immunogold studies revealed a low level of host molecules to be associated mainly with the microthrix spines of the tegument with less gold labelling being observed on the microthrix shafts.

[The biological cycle of Caryophyllaeus brachycollis Janiszweska, 1953 (Cestoda carophyllaeidae) a parasite of Barbus meridionalis Risso, 1826 in the south of France]. / Cycle biologique de Caryophyllaeus brachycollis Janiszweska, 1953 (Cestoda caryophyllaeidae) parasite de Barbus meridionalis Risso, 1826 dans le sud de la France

Caryophyllaeus brachycollis Janiszweska, 1953 has been found for the first time in France in Barbus meridionalis Risso, 1926. Experimentally, the procercoid develops in Tubifex sp. and never in Limnodrilus sp. The different possibilities of life cycle of C. brachycollis in Europe have been compared.

Distinguishing lipid pseudomembranes from larval cestodes by morphologic and histochemical means.

BACKGROUND: Contributors regularly submit specimens to our institute and suggest a diagnosis of cestode infection, but the structures in question are actually lipomembranous changes that produce lipid pseudomembranes. This required a reproducible method to distinguish lipid pseudomembranes from body walls of cestodes. METHODS: We describe and compare the morphologic and histochemical features of specimens from 20 patients. Nine specimens represented lipid pseudomembranes, and 11 represented one of the following five entities: cysticercus, coenurus, sparganum, hydatid of Echinococcus granulosus, or metastatic solid-bodied cyclophyllidean (possibly cysticercoid) larval cestodes. Specimens were stained with hematoxylin-eosin, Gomori's methenamine-silver, and 72-hour oil red O. Nine patients with cestodes, and all with lipid pseudomembranes, presented with subcutaneous lesions. RESULTS: In all specimens, oil red O provided marked contrast between lipid pseudomembranes and surrounding tissue, but focal or minimal contrast between larval cestodes and surrounding tissue. Unlike hematoxylin-eosin, Gomori's methenamine-silver stain produced distinctly different staining patterns in larval cestodes and lipid pseudomembranes. CONCLUSIONS: This technique readily permitted a simple, reproducible, and accurate distinction between lipid pseudomembranes and cestode body walls and distinguished between body walls of different cestodes.

A simple method of obtaining an enriched fraction of tegumental brush border from Hymenolepis diminuta.

A method for isolating an enriched preparation of tegumental brush border from the tapeworm, Hymenolepis diminuta, is described. Combining incubation of whole tapeworms in Krebs-Ringer/tris-maleate solution containing a hemolytic saponin, low shear-force agitation, and differential centrifugation, a pellet is obtained at 2,500 g which contains a significant concentration of surface brush border. The content of brush border in this fraction is identified by the presence of numerous microvilli, increased specific radioactivity after surface tagging with 3H-Concanavalin A, and relatively little mitochondrial contamination (succinic dehydrogenase). Based on morphological criteria, fractions sedimenting with greater force contain dense vesicles and mitochondria from the outer portion of the tegument.

Lipids from subcellular fractions of the tegument of Hymenolepis diminuta.

Lipids comprised 37% and 22.1%, respectively, of the day weights of brush border- and vesicle-rich fractions separated by differential centrifugation of isolated H. diminuta tegument. Neutral lipids of both fractions were rich in cholesterol, but also contained small amounts of glycerides, sterol esters, and (in brush borders) free fatty acids. Phosphatidyl ethanolamine was the most prevalent polar lipid in both fractions, and was particularly abundant (63.4% of total polar lipids) in vesicles; sphingomyelin, not previously reported from H. diminuta, was also present. Polar lipids of both tegumental fractions resembled each other but differed from whole worm polar lipids in fatty acid composition. Tegumental polar lipids contained lower levels of long-chain, polyunsaturated fatty acids than reported for corresponding lipids of whole worms.

Distribution and ultrastructure of two types of scolex gland cells in adult Proteocephalus macrocephalus (Cestoda, Proteocephalidea).

In the scolex-neck region of the adult Proteocephalus macrocephalus two types of eccrine gland cells are present. The first type of gland cells, localized in the frontal part of the scolex only, contains large, more or less round electron lucid granules. The second type of unicellular glands produces large electron dense granules. These electron dense granular gland cells are localized primarily in the neck region, only few are present in the scolex apex. The secretion of both types of gland cells is concentrated in the ducts opening to the exterior. The ducts are fixed to the plasmalemma of the tegument by septate junctions. The function of both types of gland cells is discussed.