Morphological observation and molecular phylogeny of Spirometra decipiens complex 1 (Cestoda: Diphyllobothriidae) found in cat from Chile.

The systematics of tapeworms in the genus Spirometra has been progressing with the accumulation of molecular genetics data, but the taxonomic status of many nominal species remains under debate. We report morphological and molecular-phylogenetic data for a Spirometra species collected from a domestic cat (Felis silvestris catus) in Chiloé Island, Chile. The Spirometra species was shown to be genetically conspecific with Spirometra decipiens complex 1 found in a Pampas fox (Lycalopex gymnocercus) from Argentina, and was closely related to a Hoary fox (Lycalopex vetulus) and rattlesnake (Crotalus durissus) from Brazil. Therefore, the presence of S. decipiens complex 1 was molecularly confirmed for the first time in Chile. The findings of the present study add useful information for the systematics of poorly known Spirometra species in South America.

Spirometra decipiens (Cestoda: Diphyllobothriidae) Collected in A Heavily Infected Stray Cat from the Republic of Korea.

Morphological and molecular characteristics of spirometrid tapeworms, Spirometra decipiens, were studied, which were recovered from a heavily infected stray cat road-killed in Eumseong-gun, Chungcheongbuk-do (Province), the Republic of Korea (=Korea). A total of 134 scolices and many broken immature and mature proglottids of Spirometra tapeworms were collected from the small intestine of the cat. Morphological observations were based on 116 specimens. The scolex was 22.8-32.6 mm (27.4 mm in average) in length and small spoon-shape with 2 distinct bothria. The uterus was coiled 3-4 times, the end of the uterus was ball-shaped, and the vaginal aperture shaped as a crescent moon was closer to the cirrus aperture than to the uterine aperture. PCR amplification and direct sequencing of the cox1 target fragment (377 bp in length and corresponding to positions 769-1,146 bp of the cox1 gene) were performed using total genomic DNA extracted from 134 specimens. The cox1 sequences (377 bp) of the specimens showed 99.0% similarity to the reference sequence of S. decipiens and 89.3% similarity to the reference sequence of S. erinaceieuropaei. In the present study, we report a stray cat heavily infected with S. decipiens identified by mitochondrial cox1 sequence analysis and morphological examinations of the adult worms.

[Effects of Omphalia lapidescens and Praziquantel on the Infectivity and Ultrastructure of Spirometra erinacei Plerocercoids].

OBJECTIVE: To study the effects of Omphalia lapidescens and praziquantel on the infectivity and ultrastructure of Spironetra erinacei plerocercoids. METHODS: The plerocercoids were taken from frogs (Rana nigromaculata). A total of 168 mice were divided into 21 groups (8 mice per group), each of them was orally infected with 5 plerocercoids. The mice in group 1-9 were inoculated with plerocercoids cultured in media respectively containing different concentrations of O. lapidescens suspension (20, 40 or 80 mg/ml) for 4, 12 or 24 h, respectively. The mice in group 10-18 were inoculated with plerocercoids cultured in media respectively containing different concentrations of praziquantel (20, 80 or 320 µg/ml) for 4, 12 or 24 h, respectively. The mice in group 19-21 were inoculated with plerocercoids cultured in normal culture fluid for 4, 12 or 24 h, respectively, and served as controls. One week after infection, the mice were sacrificed to collect the plerocercoids. Worm reduction rate was calculated. The ultrastructure changes of plerocercoids were observed under transmission electron microscope (TEM) and scanning electron microscope (SEM), respectively. RESULTS: The average number of plerocercoids detected from mice infected by pleroceroids treated with 40, 80 mg/ml O. lapidescens suspension for 4, 12 or 24 h were 1.6, 1.0, and 0.3; 0.3, 0, and 0, respectively, and significantly lower than that of the infected controls (4.1, 3.5 and 3.3) (P < 0.05); the worm reduction rates were 60.0%, 71.4%, and 90.1%; 92.7%, 100%, and 100%, respectively. The average number of pleroceroids detected from mice infected with pleroceroids treated with 320 µg/ml praziquantel for 4, 12, or 24 h were 1.9, 1.3, and 0.4, and significantly lower than that of the infected controls (P < 0.05); the worm reduction rates were 53.7%, 62.9%, and 87.9%, and lower than that of 20 µg/ml praziquantel group (14.6%, 2.9%, and 6.1%) and 80 µg/ml praziquantel group (24.4%, 17.1%, and 24.2%) (P < 0.05). The ultrastructure of plerocercoids cultured in 20 mg/ml O. lapidescens suspension, 20 or 80 µg/ml praziquantel for 4, 12 or 24 h had no significant difference compared with control groups. The plerocercoids cultured in 40 mg/ml O. lapidescens for 4 h or 320 µg/ml praziquantel for 4 or 12 h, showed mild contracture. The pleroceroids cultured in 40 mg/ml O. lapidescens for 12-24 h showed: agglutinate, fusion, fracture or abscission of microtriches, breakdown of plasma membrane, excretion of calcareous corpuscles, and tegument tissue damages. After cultured in 80 mg/ml of O. lapidescens for 24 h, the tissues of plerocercoid were damaged seriously. After cultured in 320 µg/ml praziquantel for 24 h, the plerocercoids showed: obvious contracture in the anterior end of plerocercoid, edema and bulge of plasma membrane, morphological changes of calcareous corpuscles, increase of secretory granules, glycogen depletion, and chromatin compaction in flame cells. CONCLUSION: The infectivity of Spironetra erinacei plerocercoids decreases along with the time of culture and the increase of drug concentration. Omphalia lapidescens and praziquantel can cause extensive tissue damage to the plerocercoids in vitro, and the effect of 0. lapidescens on the infectivity and ultrastructure of plerocercoid is more considerable than that of praziquantel.

Identification and characterization of a mitochondrial manganese superoxide dismutase of Spirometra erinacei.

A gene encoding the manganese superoxide dismutase (Mn-SOD) of Spirometra erinacei was identified, and the biochemical properties of the recombinant enzyme were partially characterized. The S. erinacei Mn-SOD gene consisted of 669 bp, which encoded 222 amino acids. A sequence analysis of the gene showed that it had typical molecular structures, including characteristic metal-binding residues and motifs that were conserved in Mn-SODs. An analysis of the N-terminal presequence of S. erinacei Mn-SOD revealed that it had physiochemical characteristics commonly found in mitochondria-targeting sequences and predicted that the enzyme is located in the mitochondria. A biochemical analysis also revealed that the enzyme is a typical Mn-SOD. The enzyme was consistently expressed in both S. erinacei plerocercoid larvae and adult worms. Our results collectively suggested that S. erinacei Mn-SOD is a typical mitochondrial Mn-SOD and may play an important role in parasite physiology, detoxifying excess superoxide radicals generated in the mitochondria.

[Observation on the ultrastructure of Spirometra mansoni plerocercoid].

Plerocercoids of Spirometra mansoni were collected from muscles of the frogs. Specimens were treated following the routine procedure, embedded, sliced and stained. The ultrastructure of plerocercoid was observed with transmission electron microscopy. It was found that the wall of plerocercoid consisted of tegument and parenchyma. Thornshape microtriches distributed over the outer surface of the tegument. Matrix zone had a lot of granular discoidal bodies, vesicles, mitochondria and endoplasmic reticulum. Most of the mitochondria were near the basal membrane. Parenchyma zone consisted of muscular layer, tegument cells, parenchymal cells, excretory system, and so on. Many cytoplasmic pathways of tegumentary cells stretch into muscular layer, suggesting that the tegument may be the absorptive site of nutrients.

Purification and localization of a 10 kDa calcareous corpuscle binding protein of Spirometra mansoni plerocercoid.

In cestode parasites, calcareous corpuscles are thought to be associated with a number of intracellular physiologies by regulating the trafficking of mineral components. We previously separated these particular components by Ficoll, and their binding proteins of 10 kDa and 35 kDa in the Spirometra mansoni plerocercoid (sparganum). In the present study, we purified a 10 kDa protein employing octyl-Sepharose CL-4B affinity chromatography. Anti-serum raised against the purified protein showed specific reactions to the calcareous corpuscles of the worm section by immunohistochemistry, and recognized the 10 kDa protein by immunoblotting.

Ultrastructure studies on the papillae and the nonciliated sensory receptors of adult Spirometra erinacei (Cestoda, Pseudophyllidea).

The small numerous papillae on the ventral surface of the gravid proglottid of adult Spirometra erinacei were studied by scanning electron microscopy. The arrangement of clumps of papillae was recognized on the surface of the central portion around the genital atrium, with lateral clumps being located above a pair of longitudinal nerve cords and marginal ones, on both sides of the proglottid. By transmission electron microscopy, two types of nonciliated sensory receptors were observed within the papillae. The type I, single receptor was embedded within a papilla. This dome-like sensory receptor contained two electron-dense collars and four rootlets surrounded by numerous thin filaments. The type II receptor was found arranged in groups in the area between the papillae, and the apical end was exposed to the external environment. This simple, club-like sensory receptor contained electron-lucent vesicles and microtubules. We believe that the papillae play an important role in cross-insemination.

Ultrastructural studies on the plerocercoid of Spirometra erinacei in experimental sparganosis.

The gland cell and the tegument of the Spirometra erinacei plerocercoid following parasite migration in hamsters were observed by electron microscopy. Gland cells released secretory granules mainly into the frontal pit during each stage of migration. The density of the perinuclear secretory granules decreased markedly just after parasite penetration through the host intestinal wall. Therefore, gland cells seem to play an essential role in penetration. During migration in the host, lipid-like droplets appeared in the subtegumental cells and tegument and increased in number and size during migration in the abdominal cavity, but they had disappeared from plerocercoids recovered from subcutaneous tissues. Myelin-like bodies also occurred in the tegument, subtegumental cells and medullary matrix; they were released directly through the tegument into the frontal pit. Discoidal bodies gathered in the distal part of the tegument, where filamentous microtriches appeared in regions from which microtriches had been peeled off during penetration.

Location of carbohydrate in the tegument of the procercoid of Spirometra mansonoides.

Carbohydrate distribution within vesicles of the tegumental cytoplasm of the procercoid of Spirometra mansonoides indicated that there are at least three morphologically and histochemically different vesicular types, but only one vesicular type was present in the subtegumental perikarya. This distribution of vesicles and the presence of morphologically intermediate forms could represent either the discontinuous, sequential synthesis of the various vesicular types or continued differentiation of vesicles observed in the synthetic regions of the perikarya once they move to the tegumental cytoplasm.