Complex insight on microanatomy of larval "human broad tapeworm" Dibothriocephalus latus (Cestoda: Diphyllobothriidea).

BACKGROUND: In Europe, the tapeworm Dibothriocephalus latus (syn. Diphyllobothrium latum) is a well-known etiological agent of human diphyllobothriosis, which spreads by the consumption of raw fish flesh infected by plerocercoids (tapeworm's larval stage). However, the process of parasite establishment in both intermediate and definitive hosts is poorly understood. This study was targeted mainly on the scolex (anterior part) of the plerocercoid of this species, which facilitates penetration of the parasite in intermediate paratenic fish hosts, and subsequently its attachment to the intestine of the definitive host. METHODS: Plerocercoids were isolated from the musculature of European perch (Perca fluviatilis) caught in Italian alpine lakes. Parasites were examined using confocal microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Immunofluorescence tagging was held on whole mount larvae. RESULTS: The organisation of the central and peripheral nervous system was captured in D. latus plerocercoids, including the ultrastructure of the nerve cells possessing large dense neurosecretory granules. Two types of nerve fibres run from the body surface toward the nerve plexus located in the parenchyma on each side of bothria. One type of these fibres was found to be serotoninergic and possessed large subtegumental nerve cell bodies. A well-developed gland apparatus, found throughout the plerocercoid parenchyma, produced heterogeneous granules with lucent core packed in a dense layer. Three different types of microtriches occurred on the scolex and body surface of plerocercoids of D. latus: (i) uncinate spinitriches; (ii) coniform spinitriches; and (iii) capilliform filitriches. Non-ciliated sensory receptors were observed between the distal cytoplasm of the tegument and the underlying musculature. CONCLUSIONS: Confocal laser scanning microscopy and electron microscopy (SEM and TEM) showed the detailed microanatomy of the nervous system in the scolex of plerocercoids, and also several differences in the larval stages compared with adult D. latus. These features, i.e. well-developed glandular system and massive hook-shaped uncinate spinitriches, are thus probably required for plerocercoids inhabiting fish hosts and also for their post-infection attachment in the human intestine.

Prostaglandins E2 and D2-regulators of host immunity in the model parasite Diphyllobothrium dendriticum: An immunocytochemical and biochemical study.

The spectrum of immunomodulating molecules produced by tapeworms is not yet well understood. The aims of this study, on the tapeworm Diphyllobothrium dendriticum, were: 1) detection and quantification of prostaglandins (PGs) E2 and D2 by high performance liquid chromatography; 2) visualization of PGE2 and PGD2 in specific cells, using methods of immunocytochemistry and confocal laser scanning microscopy; and 3) investigation of the ultrastructure of the cells potentially producing PGE2 and PGD2. The PGE2 immunoreaction (IR) was found in the apical terminals of the frontal glands and sensory organs in the tegument and in small neurons belonging to the main cords and commissures. PGE2-IR partly coincided with α-tubulin-IR. PGD2-IR occurred in the muscle fibers of longitudinal and transverse body muscles and coincided with phalloidin TRITC staining. Both PGE2 and PGD2 were found in the flame cells of the excretory system. Ultrastructural study of the tegument revealed two types of structures that potentially produce PGE2: ciliated and unciliated free nerve endings and frontal gland terminals reinforced with neurotubules. In the main nerve cords, small neurons were identified as potentially exhibiting PGE2-immunoreactivity. In homogenates of the plerocercoids, the measured content of PGE2 and PGD2 was 33.15ngmg-1 and 1.94ngmg-1 of fresh tissue weight, respectively. We found evidence of PGE2 and PGD2 in D. dendriticum parasitizing Coregonus autumnalis (fish) and proved excretion of PGE2 and PGD2 in response to C. autumnalis blood serum. Prostaglandins produced by D. dendriticum probably play a dual role: 1) PGE2 and PGD2 potentially modulate the fish antiparasitic immune response; 2) PGE2 is presumably necessary for proper development and function of the nervous system, and PGD2 can act as an antagonist against mediators causing muscle contraction.

High morphological plasticity and global geographical distribution of the Pacific broad tapeworm Adenocephalus pacificus (syn. Diphyllobothrium pacificum): molecular and morphological survey.

The most important causative agent of human diphyllobothriosis in South America, Diphyllobothrium pacificum, is transferred to the original genus Adenocephalus Nybelin, 1931; revised and redescribed on the basis of the evaluation of an extensive material collected mainly from northern fur seal, Callorhinus ursinus, from St. Paul Island, Alaska. Detailed analysis of morphological and morphometrical data shows a high variability in most of the characteristics traditionally used in diagnosis of diphyllobothriid tapeworms. Phylogenetic analyses based on newly characterised sequences of mitochondrial cytochrome c oxidase subunit 1 and nuclear large subunit ribosomal RNA genes consistently reveal Adenocephalus pacificus as a sister lineage to the clade formed of the remaining Diphyllobothrium species and other genera (Digramma, Diplogonoporus, Ligula). Despite the generally similar morphology, A. pacificus can be differentiated from the closely related taxa in the presence of transverse papilla-like tegumental protuberances distributed anteriorly, separated by narrow semicircular grooves on the ventral surface of proglottids between their anterior margin and the anterior edge of the male gonopore, and relatively small eggs. A. pacificus displays a relatively low host specificity (found in 9 of 16 otariids, and in accidental hosts such as man, dog and jackal, the latter representing a new host) and a uniquely wide geographical distribution on both hemispheres. In addition, suitability of morphological criteria used in diagnostics of diphyllobothriid cestodes is discussed.

First study of vitellogenesis of the broad fish tapeworm Diphyllobothrium latum (Cestoda, Diphyllobothriidea), a human parasite with extreme fecundity.

In the present study, the process of vitellogenesis of one of the most prolific organisms, the broad tapeworm, Diphyllobothrium latum, the causative agent of human diphyllobothriosis, was studied for the first time using transmission electron microscopy. Cytochemical staining with periodic acid-thiosemicarbazide-silver proteinate for detection of glycogen was applied. Starting from the periphery toward the center of the vitelline follicle four stages of vitellocytes are differentiated: immature vitellocytes, early maturing vitellocytes, advanced maturing and mature vitellocytes. Differentiation into mature vitellocytes involves the formation of shell globule clusters containing shell globules, large amount of saturated lipid droplets and glycogen. A peculiar ultrastructural feature of D. latum vitellogenesis is the presence of lamellar bodies in the cytoplasm of mature vitellocytes. This feature is similar to that present in the closely related caryophyllideans and spathebothriideans. Despite the great similarity observed in the embryonic development of diphylobothriideans, caryophyllideans and spathebothriideans, and the fact that their vitellocytes share a feature not reported from other cestode groups, there are substantial differences in the morphology of vitelline clusters, types, amount and localization of their nutritive reserves.

GABA in the nervous system of the cestodes Diphyllobothrium dendriticum (Diphyllobothriidea) and Caryophyllaeus laticeps (Caryophyllidea), with comparative analysis of muscle innervation.

The nervous system (NS) of the cestodes Diphyllobothrium dendriticum (Diphyllobothriidea) and Caryophyllaeus laticeps (Caryophyllidea) was investigated using immunocytochemistry. The GABA neurotransmitter was identified in the NS of both species; GABAergic neurons were detected in the main nerve cords (MC). GABA-like immunoreactive neurons were predominantly unipolar and exhibited more intensive immunoreactivity in the neurite than in the perikaryon. In C. laticeps , GABA-like immunoreactive somas are located in both the MCs and peripheral NS near the longitudinal muscles. The innervation of the body musculature was studied using a combination of antibodies against GABA, serotonin (5-HT), and FMRFamide and with complementary staining of F-actin. In both species, the location of GABAergic neurites is associated with all muscle layers including the subtegumental, longitudinal, transverse, and dorsoventral muscles. The cytomorphology of 5-HTergic motoneurons in the MCs of both species is described and differences in muscle innervation between D. dendriticum and C. laticeps are demonstrated. No evidence for co-localization of GABA with 5-HT or FMRFamide neurotransmitters at any particular neuron was found. Neuropiles in MCs and peripheral NS had separate sets of immunoreactive neurites. A functional role for GABA in muscle innervation is discussed.

[Molecular identification of human Diphyllobothrium nihonkaiense using mitochondrial cytochrome c oxidase subunit 1 (cox1) gene sequence].

Identification of Diphyllobothrium species has been carried out based on their morphology, especially sexual organs. In addition to these criteria, PCR-based identification methods have been developed recently. A 20 year-old Japanese living in Kochi Prefecture passed tapeworm. He was successfully treated with single dose of gastrografin. We examined the morphologic features of the proglottids and eggs using histology and scanning electron microscope. We also analyzed mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of the proglottids. The causative tapeworm species was identified as D. nihonkaiense based on the results of morphologic features and genetic analysis. We discussed the advantage of PCR-based identification methods of Diphyllobothrium species using cox1 sequence in the clinical laboratory.

Mitochondrial DNA divergence in populations of the tapeworm Diphyllobothrium nihonkaiense and its phylogenetic relationship with Diphyllobothrium klebanovskii.

Diphyllobothrium nihonkaiense [Y. Yamane, H. Kamo, G. Bylund, J.P. Wilkgren. Diphyllobothrium nihonkaiense sp. nov (Cestoda: Diphyllobothriidae)- revised identification of Japanese broad tapeworm. Shimane J Med Sci 1986;10:29-48.] and Diphyllobothrium klebanovskii [I.V. Muratov, P.S. Posokhov. Causative agent of human diphyllobothriasis - Diphyllobothrium klebanovskii sp. n. Parazitologiia. 1988;22:165-170.] are two major species of human diphyllobothriasis in Japan and Far East Russia, respectively, but their taxonomical relationship remains unclear. In this study, we analysed the DNA sequences of 16 clinical isolates of D. nihonkaiense from Japanese people, 3 isolates of D. klebanovskii from a bear in Kamchatka, and 4 clinical isolates of D. klebanovskii from native Udygeyci people in Russia, as well as 4 plerocercoids from Oncorhynchus spp. 18S rDNA and internal transcribed spacer 1 (ITS1) sequences from D. nihonkaiense and D. klebanovskii showed a high level of similarity, indicating synonymy of the two species. Analyses of mitochondrial DNA (mtDNA) sequence polymorphisms in the cox1 and nad3 genes of D. nihonkaiense (D. klebanovskii) revealed two deeply divergent lineages, A and B, with genetic distances (Kimura-2 parameter) of 0.018-0.022. Furthermore, the distinct monophyletic groupings of cox1 haplotypes corresponded to the distinct monophyletic groupings of nad3 haplotypes. The two lineages were neither distinguished by morphological features nor defined by the localities of the samples. These results suggest that the two morphologically cryptic lineages have diverged and coexisted over a long period of time.

Difilobotriosis humana: primer caso por consumo de sushi en Buenos Aires, Argentina / Human diphyllobothriosis: first case by consumption of sushi in Buenos Aires, Argentina

La difilobotriosis es una parasitosis intestinal causada por cestodes del género Diphy-llobothrium. Son consideradas áreas endémicas de esta parasitosis Escandinavia, zonas de los Grandes Lagos de América del Norte, la Patagonia Andina, entre otras. Este sería, hasta el presente, el primer informe de un caso de difilobotriosis por consumo de sushi en Buenos Aires, Argentina.

Spermatological characters in Diphyllobothrium latum (Cestoda, Pseudophyllidea).

Spermiogenesis and the ultrastructural features of the spermatozoon of Diphyllobothrium latum (Cestoda, Pseudophyllidea) are described using transmission electron microscopy. Spermiogenesis is characterized by the development of two flagella of unequal length that grow asynchronously. When the first growing flagellum starts to rotate, the second one develops. Flagellar rotation is thus asymmetric and asynchronic. It is followed by proximodistal fusion with the median cytoplasmic process. Electron-dense material is present in the apical region of the zone of differentiation in the early stages of spermiogenesis. The intercentriolar body consists of seven plates: three are electron-dense. Four attachment zones occur in the median cytoplasmic process. An atypical arrangement of striated roots was occasionally observed. The mature spermatozoon possesses two axonemes of 9 + "1" trepaxonematan pattern, nucleus, cortical microtubules, electron-dense granules, and lacks mitochondria. The ultrastructure of the anterior extremity of the spermatozoon in D. latum clearly differs from that in the bothriocephalid pseudophyllideans, mainly in the absence of a crested body and a ring of electron-dense tubular structures. The spermatological data support the assumption that the order Pseudophyllidea is formed by two unrelated clades, "Bothriocephalidea" and "Diphyllobothriidea."

Diphyllobothriasis, Brazil.

Cases of human diphyllobothriasis have been reported worldwide. Only 1 case in Brazil was diagnosed by our institution from January 1998 to December 2003. By comparison, 18 cases were diagnosed from March 2004 to January 2005. All patients who became infected ate raw fish in sushi or sashimi.

Diphyllobothrium: Neolithic parasite?

During paleoparasitological analyses on several Neolithic sites in Switzerland (Arbon-Bleiche 3) and southwestern Germany (Hornstaad-Hörnle I, Torwiesen II, and Seekirch-Stockwiesen), numerous eggs of Diphyllobothrium sp. were recovered. This is one of the earliest occurrences of this parasite during the prehistoric period in the Old World. The prevalence of this helminth in the samples studied raises the question as to how important parasitic diseases were during the Neolithic period and what their actual consequences were.

[Ultrastructure of genital system ducts of Diphyllobothrium latum (Cestoda:Pseudophyllidae): the ducts of the female reproductive system]. / Ul'trastruktura protokov polovoi sistemy Diphyllobothrium latum (Cestoda: Rseudophyllidea): protoki zhenskikh reproduktivnykh organov.

The components of the female reproductive system of Diphyllobothrium latum, including ovary, ovicapt, oviduct, vitelline ducts, vitelline reservoir, vagina, seminal receptacle, ootype with unicellular Mehlis's gland, ootype-uterine duct and uterus were observed with the electron microscope. The epithelium of the female reproductive system ducts consists of a nucleate syncytial layer. Structural differences in apical surface of the ducts, the number of nuclei and organoids in syncytial layer as well as the number of underlaid muscles were revealed. The regional differentiations of the uterus wall were registered. The middle and distal region of uterus was covered with microtriches. The filamentous microtriches were observed in apical surface of vagina. The epithelium of seminal receptacle and distal region of uterus were underlaided by the powerful muscle layers. The fertilization canal was revealed. It was shown that the formation of egg shells implemented by the deposit of vitelline globules in their surface in the ootype-uterine duct. Structural and functional differences of different parts of female apparatus in various groups of cestodes are conditioned by species biology.

[The action of fenasal on the cestode Diphyllobothrium latum]. / Vozdeistvie fenasala na tsetodu Diphyllobothrium latum.

The present study used an experimental invasion model to examine the invasion and behaviour of D.latum cestoda and its tissue structural disorders after in vivo phenasal administration. Phenasal caused an increased development of the granular endoplasmic reticulum in the cells of the tegument and yolk glands, their local spread, and a formation of vacuoles in the tegumental cells and an appearance of myelins which were rather great in size. There were no changes in tegumental microtrichia.

[Five human cases of Diphyllobothrium latum infection through eating raw flesh of redlip mullet, Liza haematocheila].

A total of 32 human diphyllobothriasis cases have been reported so far in Korea, excluding 21 egg-positive cases from stool examinations. Authors experienced five more human cases of Diphyllobothrium latum infection, especially infected due to eating raw flesh of redlip mullet, Liza haematocheila. Five cases were neighbors residing in the Puchon area, Kyonggi-do, who ate raw mullets (L. haematocheila) in a party in February 1996. The mullets were purchased at the Noryangjin fisheries market in Seoul. All of cases (2 males and 3 females) were 35 to 43 years old and healthy with the body weight range of 56-62 kg. They complained about gastrointestinal trouble and abdominal discomfort, but were in normal ranges of their hematology and urinalysis data. None revealed any sign of anemia. The patients experienced natural discharge of a chain of segments before, and showed diphyllobothriid eggs in their stool specimens when they visited our laboratory. They were administered with praziquantel (15 mg/kg of body weight) and 30 g of magnesium sulfate as a purgative. Two whole worms with the scolices (310-340 cm in length; 8-13 mm in width) were expelled each from two out of five cases after anthelmintic treatment, and the others expelled the parts of strobilae without scolex. The worms were identified as D. latum, based on the following biological characters: external morphologies, coiling of uterus, the number of uterine loops, position of genital opening, morphologies of cirrus, cirrus sac and seminal vesicle on the histological sections, position of vagina and uterine pore, and microscopical and SEM morphologies of the eggs.

Sequential development of the immune response in rainbow trout [Oncorhynchus mykiss (Walbaum, 1792)] to experimental plerocercoid infections of Diphyllobothrium dendriticum (Nitzsch, 1824).

Development of the inflammatory response of rainbow trout to experimental infections with Diphyllobothrium dendriticum plerocercoids is described using light and electron microscopy. The cellular response to plerocercoids occurred within 2 weeks post-infection (p.i.). This was followed by an increase in leucocyte numbers during weeks 3-6 p.i., with full encapsulation of plerocercoids by week 6 p.i. Neutrophils were the first leucocytes to engage the developing plerocercoid, followed by large influxes of macrophages which transformed into epithelioid cells. With longer times p.i. the accumulation of different leucocyte types increased, and a blood vascular network developed. Full development of the composite cyst was characterized by fibroplasia, particularly at the periphery of the cyst, and the subsequent deposition of a collagenous tissue matrix. Enzyme-linked immunosorbent assay (ELISA) examination of serum samples taken over the 20 week period showed that specific anti-D. dendriticum antibody titres were first detected at 5 weeks p.i. and increased to a maximum by 11 weeks p.i.

[The action of albendazole on the broad tapeworm]. / Deistvie al'bendazola na lentesa shirokogo.

Studies of the effects of the anthelminthic agent albendazole on the tissues of D. latum in vivo showed large shapeless protrusions of the outer tegumental cytoplasm on the surface of strobila, the mitochondrial degeneration in the tegumental cells and their nucleolar material segregation. After in vitro treatment cavitary formations in the outer tegumental syncytium, muscular fascicle loosening, decreased glycogen levels, and altered secret granules in the vitellaria were found. Thus, albendazole might affect the biochemical system of the parasite, since there is a relationship between some biochemical processes and morphological changes.

Scanning electron microscopic study of four Diphyllobothrium species.

Three-dimensional observation was carried out on plerocercoids and adults of Diphyllobothrium dendriticum, D. ditremum, D. latum, and D. vogeli using scanning electron microscopy. The species-specific differences between plerocercoids were recognized in the shapes of the whole body, scolex, and bothrium and the wrinkle pattern on the body surface. The differences between adult worms were also observed in the shapes of the scolex, neck, and genital papillae around the genital pore and the pattern on the egg surface. The significance of species specificity in the three-dimensional morphology of diphyllobothriid cestodes is briefly discussed.

The effect of praziquantel, an antihelminthicum, on the peptidergic nervous system of the tapeworm Diphyllobothrium dendriticum.

The effect of the antihelminthic substance Praziquantel on the peptidergic nervous system in the adult gull-tapeworm Diphyllobothrium dendriticum was tested. As well as increasing muscle contraction the drug provoked a fall in the amount and size of the peptidergic nerve terminals in the subtegumental region. The number of small clear vesicles in the terminals also fell.

[Ultrastructural changes of the tegument on Diphyllobothrium erinacei and Hymenolepis nana treated with paromomycin sulfate in vitro].

The effects of paromomycin sulfate on Diphyllobothrium erinacei and Hymenolepis nana in vitro were examined morphologically with a scanning and a transmission electron microscope. D. erinacei was incubated for 3 and 6 hours at 37 degrees C in a culture medium, 0.85% physiological saline solution, containing 0.5% paromomycin sulfate. H. nana was incubated in the same medium for 3 hours only. The concentration of paromomycin sulfate was set basing on the results which Kitamoto (1968) reported as the concentration level in feces after administrations of this drug in a clinical survey. The effect of the drug on the surface structure in both worms appeared markedly in the neck region. Mechanisms of breakdown on the tegument were supposed as follows. First, microtriches were disconnected from the tegumental surface and many vesicles were formed in the cytoplasm of the tegument. Finally, the tegument layer was excoriated to exposed the basal lamina. In 6 hours incubation, this surface of the worm suffered more damage than that in 3 hours. The damage of the basal lamina as in the case of D. latum expelled from a man by paromomycin (Y. Tongu et al.), however, could not be observed in the present study in vitro. It suggests that the destruction of basal lamina usually observed with the expelled worms from clinically treated human might be due to the combined effect of digestive enzymes secreted from host and the mechanical impact of intestinal peristalsis. Some of the vesicles in the tegument may originate from mitochondria because the fine structure of cristae were occasionally observed remaining in the vesicles.