Proteomic and Immunological Identification of Diagnostic Antigens from Spirometra erinaceieuropaei Plerocercoid

Human sparganosis is a food-borne parasitic disease caused by the plerocercoids of Spirometra species. Clinical diagnosis of sparganosis is crucial for effective treatment, thus it is important to identify sensitive and specific antigens of plerocercoids. The aim of the current study was to identify and characterize the immunogenic proteins of Spirometra erinaceieuropaei plerocercoids that were recognized by patient sera. Crude soluble extract of the plerocercoids were separated using 2-dimensional gel electrophoresis coupled with immunoblot and mass spectrometry analysis. Based on immunoblotting patterns and mass spectrometry results, 8 antigenic proteins were identified from the plerocercoid. Among the proteins, cysteine protease protein might be developed as an antigen for diagnosis of sparganosis.     

A glycoprotein from Spirometra erinaceieuropaei plerocercoids suppresses osteoclastogenesis and proinflammatory cytokine gene expression

Various parasites modify the immune-reactions of the host. We have previously shown that crude excretory/secretory (ES) products from plerocercoids of Spirometra erinaceieuropaei, the plerocercoids of which cause sparganosis in humans, suppress the expression of tumor necrosis factor (TNF)-alpha and IL-1beta in lipopolysaccharide (LPS)-stimulated macrophages. As osteoclasts are cells of the monocyte/macrophage lineage, we hypothesised that ES products might suppress receptor activator of nuclear factor kappaB ligand-induced osteoclastogenesis. Crude ES products from plerocercoids suppressed osteoclastogenesis, judged by tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cell counting, and the mature osteoclast-specific gene expression (calcitonin receptor and TRAP). Second, we purified the inhibitory factor for osteoclastogenesis from the crude ES products. The factor was a trypsin-sensitive glycoprotein and had a relative molecular mass of 90 kDa. The glycoprotein, plerocercoid-immunosuppressive factor, from crude ES products could suppress the gene expression of TNF-alpha, IL-1beta and NO synthesis in LPS-stimulated RAW264.7 macrophages.     

Evidence of the growth factor in mouse serum infected with Spirometra erinacei plerocercoids

To investigate the action of the growth factor secreted by Spirometra erinacei plerocercoids, various organ weights, body weight and head-body length were measured in Snell normal and dwarf mice after injection with the serum from mice and rats. Serum from mice infected with the plerocercoids caused significant increases in the weights of the liver and spleen, in the same manner as mice infected with the plerocercoids. However, serum from rats infected with plerocercoids did not cause significant changes in these parameters. The growth factor in the serum of mice infected with plerocercoids was stable at -20 degrees C for at least 6 months and easily passed through the peritoneum.     

Tetraphyllidean plerocercoids from Western Mediterranean cetaceans and other marine mammals around the world: a comprehensive morphological analysis

Tetraphyllidean plerocercoids have occasionally been reported in marine mammals, but they have rarely been described in detail, and the ecological significance of these infections is unclear. We described plerocercoids collected from the mucosa of the terminal colon and rectum, the anal crypts, and the hepatopancreatic ducts of 7 striped dolphins Stenella coeruleoalba, 1 Cuvier's beaked whale Ziphius cavirostris, and 3 Risso's dolphins Grampus griseus from the Spanish Mediterranean. We also examined undescribed plerocercoids from 3 cetacean species from the Atlantic and the Pacific. All plerocercoids had a lanceolate body, and a scolex with an apical sucker and 4 sessile monolocular bothridia. The bothridia had free posterior edges and an accessory sucker at their anterior end. Under light microscopy, the bothridia of some Mediterranean specimens looked bilocular without accessory suckers, but a true accessory sucker was observed in histological sections. A principal component analysis revealed 2 stable clusters of specimens along the first principal component regardless of host species. These "large" and "small" morphotypes are thought to represent early migratory stages of Phyllobothrium delphini and Monorygma grimaldii. The similarity in scolex morphology, the observation of plerocercoids buried in intestinal regions close to the sites where M. grimaldii and P. delphini occur, and the coexistence of all larval forms in the same individual hosts would support this hypothesis. Future molecular analysis may confirm it.     

Lipid catabolism in the plerocercoids of Schistocephalus solidus (Cestoda: Pseudophyllidea)

All of the enzymes of the β-oxidation sequence have been demonstrated in the plerocercoids ofS. solidus. However, the plerocercoids could not oxidize exogenous [¹⁴C-U-]palmitate although labelled palmitate was readily taken up and encorporated into the neutral and phospholipid fractions. This suggests that despite the presence of all of the enzymes of β-oxidation, the pathway is not functional in S. solidus plerocercoids; possible roles of the β-oxidation enzymes in the metabolism of S. solidus are discussed.     

The effects of a change in the growth rate of roach, Rutilus rutilus (L.), on the biology of the fish tapeworm Ligula intestinalis (L.)

Investigations into the biology of the roach and the pathogenic tapeworm Ligula intestinalis (L.) populations at Slapton Ley, Devon were carried out between October 1982 and December 1984. Data collected from the lake since 1977 have also been re-analysed to determine how the dramatic improvement in the individual growth rate of the roach over this period may have affected the growth, maturation and life-cycle of Ligula. Only the very young roach at this site become infected, so it was possible to follow cohorts of plerocercoids of similar age through each roach year class. Results for the 1978 and 1983 year classes are presented in detail. The roach grew extremely rapidly from May to August in each year, resulting in a pronounced cyclical pattern of changes in the condition of the roach, with the lowest condition occurring in late winter and spring. The yearly increase in the roach growth rate was accompanied by an increase in plerocercoid growth rate in the 0 + roach, but not in the 1 + roach. The growth rate of the plerocercoids was very high compared to that at other sites. It is usual for the parasite index (PI) of Ligula-infected fish to be high and to increase throughout their first few years of life. At Slapton, however, a lack of multiple infections has prevented high PIs from occurring, and in recent years the growth rate of the roach has been so high that the plerocercoids were unable to maintain a high weight relative to the fish, and the highest PIs occurred in the 0 + roach throughout late winter and spring. In recent year classes, therefore, the maximum PIs and highest pathogenicities coincided with the period of lowest condition in the 0+ roach. Observations of both caged and natural populations of 0+ roach over winter showed that a significant loss of roach containing the larger plerocercoids occurred from the population. In vitro cultivation of Ligula plerocercoids showed that they were capable of maturation at weights of 0.5 g, and only 6 months after having infected the roach. The increase of the growth rate of the plerocercoids in the 0+ roach has therefore resulted in a greater proportion of these plerocercoids being capable of infecting the definitive host. As a result of the increase in individual growth rate of the roach at Slapton, the potential for Ligula transmission, as measured in terms of both their pathogenicity and maturity, has shifted from the 1 + to the 0+ roach.     

Electron microscopy of the separated outer tegument of the sparganum and its antigenicity

The author reported previously on separation of the outer tegument of the spargana (plerocercoids of Spirometra mansoni) using high concentration of urea solution. To determine which layer of the tegument is separated by this method, an electron microscopic analysis has been processed in this study. It was confirmed that the basement layer of the tegument is separated from the parenchyme of the sparganum. In addition, the antigenicity of the separated outer tegument against the human sparganosis patient sera was evaluated. Numerous antigenic proteins, including 16 and 55 kDa proteins, were noticed in the separated tegument; however, there were no diagnostic 31/36 kDa molecules in this tegument. The molecules reactive with the patient sera in the tegument are to be characterized in future studies.     

The fine structure of frontal glands in adult cestodes

Kuperman B. I. & Davydov V. G. 1982. The fine structure of frontal glands in adult cestodes. International Journal for Parasitology12: 285–293. The localization and structure of frontal glands of adult cestodes of 20 species from 4 orders: Caryophyllidea, Pseudophyllidea, Proteocephalidea and Cyclophyllidea were studied by light and electron microscopy. In cestodes of various orders there was evidence of differences in the ultrastructure of secretory cells, in the character of the secretion, and in the method of its discharge. The localization of glands in most adult cestodes is in the scolex. In Cyatocephalus, Ligulidae, Bothriocephalus claviceps, they are distributed also in the anterior and middle part of the body. In the species Caryophylleus (C. laticeps) and Khawia sinensis a major gland complex previously not described was discovered. The glands of the cestodes investigated may be divided into three main groups by their structure, localization and functional role in ontogenesis: (1) penetration glands in oncospheres which develop into procercoids and plerocercoids; (2) glands of plerocercoids and adult cestodes which develop in the gut; (3) gland complexes in the representatives of the order Caryophyllidea. The secretion is usually discharged at the scolex region and less frequently on the lateral surface. Three different ways of discharging secretory material from the cestodes body are described.     

Aspects of the biology of the plerocercoid of Grillotia erinaceus (van Beneden, 1858) (Cestoda: Trypanorhyncha) in haddock Melanogrammus aeglefinus (L.)

From one to thirty Grillotia erinaceus plerocercoids were found in 3714 of 8228 haddock Melanogrammus aeglefinus caught in the northern North Sea, to the north and west of Scotland and at Faroe. Plerocercoids were also found in 30 of 64 cod from Aberdeen Bay, and in 44 of 55 saithe from the Firth of Clyde. In haddock, most plerocercoids occurred encysted along the intestine, and their frequency fitted a negative binomial distribution. Incidence and intensity of infestation increased with host age and there was no host sex difference in incidence. Three maturity stages were recognized, the proportions of which were consistent between haddock length groups. No precipitin bands developed in gel diffusion tests using live plerocercoids and sera from infested and uninfested haddock. Attempts to transfer plerocercoids between haddock were unsuccessful. Speculations are made on the possible course of the life-cycle.     

Studies on Ligula intestinalis Some aspects of the pathology in the second intermediate host

A 3 year survey of a lake, in which 120 roach, Rutilus rutilus , were examined revealed the presence of the plerocercoids of Ligula intestinalis in more than 90 % of the fish. The average number of parasites per fish was 4.2 although fish exceeding 32 g were not ligulosed. Gudgeon, Gobio gobio , of all sizes were affected. Implants of the plerocercoids into small roach, gudgeon and goldfish, Carassius auratus , were successful but not in the case of Perca fluviatilis, Esox lucius and Leuciscus cephalus. The pathological effects of the plerocercoids on roach and gudgeon are described.     

Excretory/secretory products from plerocercoids of Spirometra erinaceieuropaei suppress the TNF-alpha gene expression by reducing phosphorylation of ERK1/2 and p38 MAPK in macrophages

We previously reported that excretory/secretory products from plerocercoids of Spirometra erinaceieuropaei suppress gene expression and production of tumour necrosis factor-alpha in murine macrophages stimulated with lipopolysaccharide. The present study investigated the suppressive mechanisms of tumour necrosis factor-alpha mRNA by excretory/secretory products in lipopolysaccharide-stimulated murine macrophages. Electrophoretic mobility shift assay and supershift assay revealed that neither nuclear translocation of nuclear factor-kappa B nor conformation of the p50/p65 nuclear factor-kappa B subunits was affected by the treatment of excretory/secretory products in lipopolysaccharide-stimulated macrophages. Inhibition of extracellular signal-regulated protein kinase 1/2 with PD98059 or p38 mitogen-activated protein kinase with SB203580 partially reduced tumour necrosis factor-alpha mRNA expression, and a combination of the two inhibitors additionally suppressed the level of tumour necrosis factor-alpha mRNA, revealing that both pathways are crucial for full induction of the gene. Northern blot analysis showed that excretory/secretory products additionally suppressed tumour necrosis factor-alpha mRNA expression in cells treated with PD98059 or SB208530 and, in turn, we found that excretory/secretory products reduced phosphorylation of extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase in lipopolysaccharide-stimulated macrophages by Western blot analysis. This is the first report demonstrating that excretory/secretory products from parasites suppress tumour necrosis factor-alpha mRNA expression by reducing phosphorylation of extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase without any effect on nuclear factor-kappa B activity in macrophages stimulated with lipopolysaccharide. We hypothesise that excretory/secretory products may enable this parasite to survive within the host.     

An enzyme study of purine nucleotide biosynthesis in the plerocercoids of cestodes in the fam. Ligulidae]

By means of spectrophotometric method there was determined the activity of three enzymes of biosynthesis of purine nucleotides: amino imidazole ribonucleotide-carboxylase (AIR-carboxylase, EC 4.1.1.21), an enzyme of biosynthesis of purine nucleotides de novo in plerocercoids of Schistocephalus pungitii and Digramma interrupta; inosine monophosphate-dehydrogenase (IMPh-dehydrogenase, EC 1.2.1.14), an enzyme of salvage path, and adenylosuccinate lyase (EC 4.3.2.2), an enzyme taking part both in biosynthesis de novo and salvage in plerocercoids of Schistocephalus pungitii. The activity of AIR-carboxylase was not determined. Specific activities of adenylosuccinate lyase and IMPh-dehydrogenase amount to (1.3 +/- 0.3) x 10(-3) and (1.2 +/- 0.4) x 10(-3) mumole/min.mg protein, respectively. The activity of the three enzymes was determined in the liver of ten-spined stickleback, a host of S. pungitii plerocercoids. The question of metabolic dependence of Ligulidae plerocercoids on hosts to provide for purine bases is discussed.     

Survey of Spirometra erinaceieuropaei in frogs in Taiwan and its experimental infection in cats

Eighteen of 56 (32.1%) wild Rana limnocharis from central and south Taiwan were found to contain plerocercoids of Spirometra erinaceieuropaei. This is the first report of S. erinaceieuropaei infections in frogs in Taiwan, with the plerocercoids being recovered from the thigh and back muscles or under the skin. Other species of frogs examined, including nine wild R. latouchii, one wild Buergeria robustus and 110 cultured R. rugulosa were free of infection. The plerocercoids were orally inoculated into four cats; three of which were each given a single plerocercoid and one a dose of three plerocercoids. Daily faecal examination showed that two cats started shedding eggs of S. erinaceieuropaei on day 8 postinfection (PI) and the other two on day 10 PI. The highest eggs per gram and eggs per day for a single worm was found to be 428,000 and 14,416,000 respectively. Only the cat inoculated with three plerocercoids shed proglottids in its faeces during the 2 month observation period.     

Effect of cestodes on the tissue lipid composition of the burbot and stickleback

The effect of cestodes of the genus Diphyllobothrium on the lipoid contents of tissues of freshwater fishes has been studied. The level of general lipids in the liver of burbot and stickleback infected with plerocercoids of D. latum and D. vogeli, respectively, decreases. It occurs in general on account of decrease in contents of structural lipids, phospholipids, while the contents of triacylglycerines, cholesterol and its ethers change but negligibly. Simultaneous parasitism of plerocercoids of D. latum and Triaenophorus nodulosus in the liver of burbot causes especially great changes in the lipoid metabolism. Certain changes in the ratio of some phospholipid fractions take place in infected tissues: the decrease in the level of phosphatidylcholine and the parallel rise in the fraction of lysophosphatidylcholine. The ratio between other phospholipids virtually does not change.     

On some cestodes parasitizing freshwater fish in Italy.

The paper presents a systematic survey of some cestodes parasitizing freshwater fish in Italy. The following eight species were recorded: Monobothrium wageneri, Cyathocephalus truncatus, Triaenophorus nodulosus (plerocercoids and adults), Bothriocephalus acheilognathi, B. claviceps, Ligula intestinalis (plerocercoids), Schistocephalus sp. (plerocercoids) and Proteocephalus percae from Perca fluviatilis which is reported from freshwater fish in Italy for the first time. All the tapeworms recovered are described and figured.     

A new method for concentration of proteins in the calcareous corpuscles separated from the spargana of Spirometra erinacei

Calcareous corpuscles are a characteristic structure found in larval and adult stage cestodes. These corpuscles are known to contain several protein components and to possess protein-binding activity. However, the proteins bound to calcareous corpuscles in situ have not been studied. The present study was undertaken to identify the proteins on calcareous corpuscles. Calcareous corpuscles were purified from the plerocercoids (= spargana) of Spirometra erinacei, and serially dissolved using 0.1 M sulfamic acid solution. Collected supernatants were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. The results showed that only the fraction remaining after the 19th dissolved fraction contained proteins. A total of 20 protein molecules were detected in gel, with major bands at 56, 53, 46, 40, 35, 29, 28, 24.5, 21, 19, 16, 13, 10 and 8 kDa. In particular, the proteins corresponding to the 21 and 16 kDa bands were most abundant. Our results demonstrated for the first time the protein contents of the calcareous corpuscles of spargana. Further studies on the functions of these proteins are required.     

Differential aggregation properties of secretory proteins that are stored in exocrine secretory granules of the pancreas and parotid glands

Low-pH- and calcium-induced aggregation of regulated secretory proteins has been proposed to play a role in their retention and storage in secretory granules. However, this has not been tested for secretory proteins that are stored in the exocrine parotid secretory granules. Parotid granule matrix proteins were analyzed for aggregation in the presence or absence of calcium and in the pH range of 5.5 to 7.5. Amylase did not aggregate under these conditions, although <10% of parotid secretory protein (PSP) aggregated below pH 6.0. To test aggregation directly in isolated granules, rat parotid secretory granules were permeabilized with 0.1% saponin in the presence or absence of calcium and in the pH range of 5.0 to 8.4. In contrast to the low-pH-dependent retention of amylase in exocrine pancreatic granules, amylase was quantitatively released and most PSP was released from parotid granules under all conditions. Both proteins were completely released upon granule membrane solubilization. Thus neither amylase nor PSP show low-pH- or calcium-induced aggregation under physiological conditions in the exocrine parotid secretory granules.     

The contents of adenine nucleotides and glycolytic and tricarboxylic acid cycle intermediates in activated and non-activated plerocercoids of Schistocephalus solidus (Cestoda: Pseudophyllidea)

The steady state content of adenine nucleotides, inorganic phosphate and glycolytic and tricarboxylic acid cycle intermediates were measured in freeze clamped plerocereoids of S. solidus taken from the body cavity of infected fish (non-activated plerocercoids) and the results compared with the metabolite levels in plerocercoids which had been cultured in vitro for 12 h (activated plerocercoids). Of the glycolytic intermediates, the levels of fructose-6-phosphate, fructose-1, 6-diphosphate, 3-phosphoglycerate, phosphoenolpyruvate and lactate all decreased on activation, the remainder of the glycolytic intermediates did not alter significantly. In contrast, the levels of the tricarboxylic acid cycle intermediates all increased 2–3 fold on activation, whilst the adenine nucleotides remained virtually unchanged. The differences in the steady state content in intermediary metabolites in activated and non-activated plerocereoids are discussed in relation to possible mechanisms of metabolic control.     

Characterization of Spirometra erinaceieuropaei Plerocercoid Cysteine Protease and Potential Application for Serodiagnosis of Sparganosis

BackgroundSparganosis is a neglected but important food-borne parasitic zoonosis. Clinical diagnosis of sparganosis is difficult because there are no specific manifestations. ELISA using plerocercoid crude or excretory–secretory (ES) antigens has high sensitivity but has cross-reactions with other helminthiases. The aim of this study was to characterize Spirometra erinaceieuropaei cysteine protease (SeCP) and to evaluate its potential application for serodiagnosis of sparganosis.ConclusionsThe rSeCP had the CP enzymatic activity and SeCP seems to be important for the survival of plerocercoids in host. The rSeCP is a potential diagnostic antigen for sparganosis.