The parasite Schistocephalus solidus secretes proteins with putative host manipulation functions.

BACKGROUND: Manipulative parasites are thought to liberate molecules in their external environment, acting as manipulation factors with biological functions implicated in their host's physiological and behavioural alterations. These manipulation factors are part of a complex mixture called the secretome. While the secretomes of various parasites have been described, there is very little data for a putative manipulative parasite. It is necessary to study the molecular interaction between a manipulative parasite and its host to better understand how such alterations evolve. METHODS: Here, we used proteomics to characterize the secretome of a model cestode with a complex life cycle based on trophic transmission. We studied Schistocephalus solidus during the life stage in which behavioural changes take place in its obligatory intermediate fish host, the threespine stickleback (Gasterosteus aculeatus). We produced a novel genome sequence and assembly of S. solidus to improve protein coding gene prediction and annotation for this parasite. We then described the whole worm's proteome and its secretome during fish host infection using LC-MS/MS. RESULTS: A total of 2290 proteins were detected in the proteome of S. solidus, and 30 additional proteins were detected specifically in the secretome. We found that the secretome contains proteases, proteins with neural and immune functions, as well as proteins involved in cell communication. We detected receptor-type tyrosine-protein phosphatases, which were reported in other parasitic systems to be manipulation factors. We also detected 12 S. solidus-specific proteins in the secretome that may play important roles in host-parasite interactions. CONCLUSIONS: Our results suggest that S. solidus liberates molecules with putative host manipulation functions in the host and that many of them are species-specific.

The role of fish helminth parasites in monitoring metal pollution in aquatic ecosystems: a case study in the world's most productive platinum mining region.

Due to the increasing consumption of platinum (Pt), especially in automobile exhaust catalysts, environmental concentrations of Pt are of emerging concern worldwide. Limited information exists on environmental concentrations, particularly in Pt mining regions, while South Africa is the world's main supplier of Pt. Moreover, other metals are also released as by-products of Pt mining, which might also cause environmental concern. Certain fish parasite taxa have the ability to accumulate metals orders of magnitude higher than their hosts and can be used to reliably detect metals with naturally low abundance. Studies on Pt accumulation in parasite-host systems are limited. Therefore, the aims of the present study were (1) to determine the accumulation of a variety of metals (cadmium (Cd), chromium (Cr), copper (Cu), nickel (Ni), lead (Pb), platinum (Pt), and zinc (Zn)) in helminth fish parasites compared with their hosts from a reference site and an impoundment impacted by Pt mining activities; (2) to assess whether there is a difference between bioaccumulation of metals in infected and uninfected hosts, as well as between hosts with different infection intensities; and (3) to compare the biomarker responses (acetylcholine esterase activity (AChE), metallothionein content (MT), catalase activity (CAT), reduced glutathione content (GSH), malondialdehyde content (MDA), protein carbonyls induction (PC), superoxide dismutase activity (SOD), and cellular energy allocation (CEA)) between infected and uninfected hosts. The cestode Atractolytocestus huronensis accumulated significantly higher concentrations of Cr, Ni, and Pt than their host Cyprinus carpio, while the nematode Contracaecum sp. accumulated significantly higher concentrations of Pt and Zn than their host Clarias gariepinus. Infected fish showed lower metal concentrations compared to uninfected fish, while the parasites had no significant effects on their hosts' biomarker responses. The parasites demonstrated the bioavailability of metals derived from Pt mining activities and their ability to resist its toxic effects. Thus, these parasites are promising sensitive accumulation indicators for Cr, Ni, Pb, and Pt contaminations from Pt mining activities.

Polycyclic aromatic hydrocarbons in the cestode Oncomegas wageneri parasite of Mexican flounder Cyclopsetta chittendeni.

The concentrations of polycyclic aromatic hydrocarbon metabolites (PAHm) and their bioconcentration factors (BCF) were determined in the larval stages of the cestode Oncomegas wageneri, recovered from the intestine of the Mexican flounder Cyclopsetta chittendeni, in the southern Gulf of Mexico. The PAHm concentrations in O. wageneri were measured using fixed-wavelength fluorescence spectrometry and compared with PAHm concentrations in host bile. Oncomegas wageneri PAHm concentrations were markedly higher than those in host tissues. The highest BCF values were obtained for 1-hydroxypyrene (OHP) and benzo(a)pyrene (BaP). Using a General Linear Model, a significant negative relationship was found between O. wageneri PAHm concentrations (as response variable) and the number of O. wageneri and oil well proximity. Low BCF values and PAHm concentrations in C. chittendeni correlated positively with O. wageneri PAHm concentrations. In contrast, high BCF values for PAHm concentrations in C. chittendeni had a negative association with O. wageneri PAHm concentrations. This study provides the first evidence of the presence of PAHm in intestinal larval cestodes of marine flatfishes, demonstrating levels of PAHm that were higher than levels in their hosts.

Levels of heavy metals in the straightfin barb Enteromius paludinosus (Peters 1852) from River Malewa, Naivasha, Kenya.

There have been several studies on heavy metals in Lake Naivasha. However, none of them has reported the levels of mercury (Hg), arsenic (As), and chromium (Cr). Moreover, there are no studies on the heavy metals' concentrations in the straightfin barb (Enteromius paludinosus, Peters 1852), a fish species that hosts a parasite (Ligula intestinalis), the latter having been reported to have a high ability to absorb heavy metals from its host. This paper therefore addresses the accumulation of heavy metals, namely arsenic (As), chromium (Cr), lead (Pb), and mercury (Hg) in the tissues of straightfin barb, Enteromius paludinosus (Peters 1852) from the mouth of River Malewa in Lake Naivasha, Kenya. A total of 1307 fish were collected during the month of November 2017. Water samples, sediment samples, 25 fish muscle tissues, and its endoparasite, the cestode Ligula intestinalis, were isolated, and heavy metal concentrations were determined using the thermal-electron atomic absorption spectrophotometer at the Lake Nakuru Water Quality Testing Laboratory. The concentrations of heavy metals in the sediment were below the lowest effect level in sediment, threshold effect concentration in sediment, severe effect concentration in sediment, and the shale values of sedimentary rocks thus showing no sign of pollution. In the muscle tissues of the fish, As, Cr, Pb, and Hg showed high levels with mean concentrations of 5.0696, 22.0854, 45.2108, and 1.5458 mg/kg ww, respectively. Bioconcentration factors further supported the observation that trace element accumulation was higher in fish compared with sediment and water. The target hazard quotients of As, Cr, Pb, and Hg obtained for both the female and male were > 1 indicating a possible health risk associated with the consumption of E. paludinosus. The bioaccumulation factors (BAFs) for L. intestinalis were 2.4093, 2.1873, 5.8601, and 5.1395 for As, Cr, Pb, and Hg, respectively, indicating the potential of the cestode in the accumulation of heavy metals from the host; hence, it can be used as an accumulation bioindicator.

Report: An ex vivo up-take of levamisole molecules by cestode (Monezia expensa) of goat (Capra hirsa) and its detection through RP-HPLC.

Detection of various molecules of drugs remained a prime issue especially in tissues of animals, humans and in their target parasites. The cestode/tapeworms pose a dilemma because of their weird body composition and uptake pattern of nutrients and medicines especially through absorption by tegument. We selected levamisole; thought to be potent antiparasitic/ani-cestodal drug. The uptake of levamisole (LEV) through cestodeal tissues is studied through HPCL in this paper. High performance liquid chromatography technique has been utilized to know the uptake of levamisole in tissues of cestodes of Goat (Monezia expensa) in small ruminants. The drug was exposed to M. expensa by in vitro till its death or a parasite ceases its movement. The tissue/ part of proglattids of the M. expensa were homogenized with some modifications and levamisole extraction was performed with liquid phase extraction method. The evaporation of solvent was done and the residual cestodal tissues were cleaned by solid phase. After the solid phase extraction method, the recovery of drug, detection and quantification of levamisole from cestodal tissues was determined through Reverse Phase Column High Performance Liquid Chromatography (RP-HPLC). Levamisole (LEV) molecules assay was obtained on a C18 reverse-phase (20um, 6mm x 150mm) column at flow rate of 1ml/min using acetonitrile and ammonium acetate as mobile phase and UV detection was done at 254nm. The development of method of Levamisole (LEV) detection from cestodal tissues by HPLC in vitro samples has been demonstrated first time in Pakistan, which can provide the solution of parasitic control and provide in sight in to the uptake of anti cestodal drugs either against human or livestock parasites.

Comparative proteomics of the larval and adult stages of the model cestode parasite Mesocestoides corti.

Mesocestoides corti is a widely used model for the study of cestode biology, and its transition from the larval tetrathyridium (TT) stage to the strobilated, adult worm (ST) stage can be induced and followed in vitro. Here, a proteomic approach was used to describe and compare M. corti TT and ST protein repertories. Overall, 571 proteins were identified, 238 proteins in TT samples and 333 proteins in ST samples. Among the identified proteins, 207 proteins were shared by TTs and STs, while 157 were stage-specific, being 31 exclusive from TTs, and 126 from STs. Functional annotation revealed fundamental metabolic differences between the TT and the ST stages. TTs perform functions related mainly to basic metabolism, responsible for growth and vegetative development by asexual reproduction. STs, in contrast, perform a wider range of functions, including macromolecule biosynthetic processes, gene expression and control pathways, which may be associated to its proglottization/segmentation, sexual differentiation and more complex physiology. Furthermore, the generated results provided an extensive list of cestode proteins of interest for functional studies in M. corti. Many of these proteins are novel candidate diagnostic antigens, and/or potential targets for the development of new and more effective antihelminthic drugs. BIOLOGICAL SIGNIFICANCE: Cestodiases are parasitic diseases with serious impact on human and animal health. Efforts to develop more effective strategies for diagnosis, treatment or control of cestodiases are impaired by the still limited knowledge on many aspects of cestode biology, including the complex developmental processes that occur in the life cycles of these parasites. Mesocestoides corti is a good experimental model to study the transition from the larval to the adult stage, called strobilation, which occur in typical cestode life-cycles. The performed proteomics approach provided large-scale identification and quantification of M. corti proteins. Many stage-specific or differentially expressed proteins were detected in the larval tetrathyridium (TT) stage and in the strobilated, adult worm (ST) stage. Functional comparative analyses of the described protein repertoires shed light on function and processes associated to specific features of both stages, such as less differentiation and asexual reproduction in TTs, and proglottization/segmentation and sexual differentiation in ST. Moreover, many of the identified stage-specific proteins are useful as cestode developmental markers, and are potential targets for development of novel diagnostic methods and therapeutic drugs for cestodiases.

PCBs in fish and their cestode parasites in Lake Victoria.

Polychlorinated biphenyls (PCBs) are classified as persistent organic pollutants (POPs) regulated by the Stockholm Convention (2001). Although their production and use was stopped almost three decades ago, PCBs are environmental persistent, toxic, and bioaccumulate in biota. We assessed the levels of 7 PCB congeners (IUPAC nos. 28, 52, 101, 118, 138, 153, and 180) in sediment and fish (Oreochromis niloticus, Lates niloticus, and Rastrineobola argentea) and evaluated the potential of cestode fish endoparasite (Monobothrioides sp., Proteocephalaus sp., and Ligula intestinalis) as biomonitors of PCBs in Lake Victoria, Kenya. The median concentration of Σ7PCBs in sediments and fish were 2.2-96.3 µg/kg dw and 300-3,000 µg/kg lw, respectively. At all the sampling sites, CB138, CB153, and CB180 were the dominant PCB congeners in sediment and fish samples. Compared to the muscle of the piscine host, Proteocephalaus sp. (infecting L. niloticus) biomagnified PCBs ×6-14 while Monobothrioides sp. (infecting O. niloticus) biomagnified PCBs ×4-8. Meanwhile, L. intestinalis (infecting R. argentea) biomagnified PCBs ×8-16 compared to the muscle of unparasitized fish. We demonstrate the occurrence of moderate to high levels of PCB in sediments and fish in Lake Victoria. We also provide evidence that fish parasites bioaccumulate higher levels of PCBs than their piscine hosts and therefore provide a promising biomonitor of PCBs. We urge further a long-term study to validate the use of the above cestode fish parasites as biomonitoring tools for PCBs.

Molecular phylogeny of Cyclophyllidea (Cestoda: Eucestoda): an in-silico analysis based on mtCOI gene.

Order Cyclophyllidea (of cestode platyhelminths) has a rich diversity of parasites and includes many families and species that are known to cause serious medical condition in humans and domestic and wild animals. Despite various attempts to resolve phylogenetic relationships at the inter-family level, uncertainty remains. In order to add resolution to the existing phylogeny of the order, we generated partial mtCO1 sequences for some commonly occurring cyclophyllidean cestodes and combined them with available sequences from GenBank. Phylogeny was inferred taking a total 83 representative species spanning 8 families using Bayesian analysis. The phylogenetic tree revealed Dilepididae as the most basal taxon and showed early divergence in the phylogenetic tree. Paruterinidae, Taeniidae and Anoplocephalidae showed non-monophyletic assemblage; our result suggests that the family Paruterinidae may represent a polyphyletic group. The diverse family Taeniidae appeared in two separate clades; while one of them included all the members of the genus Echinococcus and also Versteria, the representatives of the genera Taenia and Hydatigera clubbed in the other clade. A close affinity of Dipylidiidae with Taenia and Hydatigera was seen, whereas existence of a close relationship between Mesocestoididae and Echinococcus (of Taeniidae) is also demonstrated. The crown group comprised the families Anoplocephalidae, Davaineidae, Hymenolepididae and Mesocestoididae, and also all species of the genus Echinococcus and Versteria mustelae; monophyly of these families (excepting Anolplocephalidae) and the genus Echinococcus as well as its sister-taxon relation with V. mustelae is also confirmed. Furthermore, non-monophyly of Anoplocephalidae is suggested to be correlated with divergence in the host selection.

Interactions between the intestinal cestode Polyonchobothrium clarias (Pseudophyllidea: Ptychobothriidae) from the African sharptooth catfish Clarias gariepinus and heavy metal pollutants in an aquatic environment in Egypt.

In an aquatic environment, there is a profound and inverse relationship between environmental quality and disease status of fish. Parasites are one of the most serious limiting factors in aquaculture. Therefore, the present investigation was carried out during the period of February-December 2014 to determine the parasitic infections in the African sharptooth catfish Clarias gariepinus, relative to the capability of internal parasites to accumulate heavy metals. Up to 100 catfish were examined for gastrointestinal helminths and 38% of fish were found to be infected with the cestode Polyonchobothrium clarias. The morphology of this parasite species, based on light and scanning electron microscopy, revealed that the adult worm was characterized by a rectangular scolex measuring 0.43-0.58 (0.49 ± 0.1) mm long and 0.15-0.21 (0.19 ± 0.1) mm wide, with a flat to slightly raised rostellum armed with a crown with two semicircles each bearing 13-15 hooks, followed by immature, mature and gravid proglottids which were about 29-55 (45), 16-30 (24) and 15-39 (28) in number, respectively. The mature proglottid contained a single set of genitalia in which medullary testes measured 0.09-0.13 (0.11 ± 0.01) mm long and 0.05-0.08 (0.06 ± 0.01) mm wide; a bi-lobed ovary was situated near the posterior margin of the proglottid, extending laterally up to the longitudinal excretory canals; the tubular uterus arose from the ootype up to the anterior margin of the proglottid; and vitelline follicles were cortical. The greater portion of the gravid proglottid was occupied by a uterus filled with unoperculate and embryonated eggs. Chemical analysis confirmed that the concentrations of heavy metals (Zn, Cu, Mn, Cd, Ni and Pb) accumulated in P. clarias were higher than in fish tissues and values recommended by FAO/WHO, with the exception of Zn, which was found to be higher in fish kidneys than in the cestode. This supports the hypothesis that cestodes of fish can be regarded as useful bioindicators when evaluating the environmental pollution of aquatic ecosystems by heavy metals.

Differential kinetics at PK/PEPCK branch point in the cestode, Raillietina echinobothrida.

Pyruvate kinase (PK; EC 2.7.1.40) and phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) are essential regulatory enzymes of glucose oxidation in helminths, the PK/PEPCK branch point being the first divergent step between carbohydrate catabolism of the parasites and their hosts. Recently, PEPCK from the cestode parasite, Raillietina echinobothrida, has been purified and characterized. In order to find out the differential kinetics, if any, at PK/PEPCK branch point in the parasite, in this study, we purified and characterized the parasite PK and compared it with the parasite PEPCK. The purified PK displayed standard Michaelis-Menten kinetics with Kmapp of 77.8 µM for its substrate PEP, whereas the Kmapp was 46.9 µM for PEPCK. PEP exhibited differential kinetics at PK/PEPCK branch point of the parasite and behaved as a homotropic effector for PEPCK, but not for PK. The inhibitory constant (Ki) for genistein and daidzein (phytochemicals from Flemingia vestita) was determined and discussed. From these results, we hypothesize that PK/PEPCK branch point is a probable site for anthelmintic action.

Multiple isotope analyses of the pike tapeworm Triaenophorus nodulosus reveal peculiarities in consumer-diet discrimination patterns.

Previous studies of dietary isotope discrimination have led to the general expectation that a consumer will exhibit enriched stable isotope levels relative to its diet. Parasite-host systems are specific consumer-diet pairs in which the consumer (parasite) feeds exclusively on one dietary source: host tissue. However, the small numbers of studies previously carried out on isotopic discrimination in parasite-host (ΔXP-HT) systems have yielded controversial results, showing some parasites to be isotopically depleted relative to their food source, while others are enriched or in equilibrium with their hosts. Although the mechanism for these deviations from expectations remains to be understood, possible influences of specific feeding niche or selection for only a few nutritional components by the parasite are discussed. ΔXP-HT for multiple isotopes (δ13C, δ15N, δ34S) were measured in the pike tapeworm Triaenophorus nodulosus and two of its life-cycle fish hosts, perch Perca fluviatilis and pike Esox lucius, within which T. nodulosus occupies different feeding locations. Variability in the value of ΔXP-HT calculated for the parasite and its different hosts indicates an influence of feeding location on isotopic discrimination. In perch liver ΔXP-HT was relatively more negative for all three stable isotopes. In pike gut ΔXP-HT was more positive for δ13C, as expected in conventional consumer-diet systems. For parasites feeding on pike gut, however, the δ15N and δ34S isotope values were comparable with those of the host. We discuss potential causes of these deviations from expectations, including the effect of specific parasite feeding niches, and conclude that ΔXP-HT should be critically evaluated for trophic interactions between parasite and host before general patterns are assumed.

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.

Isotopic discrimination of stable isotopes of nitrogen (δ15N) and carbon (δ13C) in a host-specific holocephalan tapeworm.

During the past decade, parasites have been considered important components of their ecosystems since they can modify food-web structures and functioning. One constraint to the inclusion of parasites in food-web models is the scarcity of available information on their feeding habits and host-parasite relationships. The stable isotope approach is suggested as a useful methodology to determine the trophic position and feeding habits of parasites. However, the isotopic approach is limited by the lack of information on the isotopic discrimination (ID) values of parasites, which is pivotal to avoiding the biased interpretation of isotopic results. In the present study we aimed to provide the first ID values of δ(15)N and δ(13)C between the gyrocotylidean tapeworm Gyrocotyle urna and its definitive host, the holocephalan Chimaera monstrosa. We also test the effect of host body size (body length and body mass) and sex of the host on the ID values. Finally, we illustrate how the trophic relationships of the fish host C. monstrosa and the tapeworm G. urna could vary relative to ID values. Similar to other studies with parasites, the ID values of the parasite-host system were negative for both isotopic values of N (Δδ(15)N = - 3.33 ± 0.63‰) and C (Δδ(13)C = - 1.32 ± 0.65‰), independent of the sex and size of the host. By comparing the specific ID obtained here with ID from other studies, we illustrate the importance of using specific ID in parasite-host systems to avoid potential errors in the interpretation of the results when surrogate values from similar systems or organisms are used.

Fish tapeworm Khawia sinensis: an indicator of environmental microcystins?

OBJECTIVES: Parasites have recently been recognized as accumulation indicators that take up and bio-concentrate substances from environmental pollution. Interestingly, helminths of fish are known to accumulate metals from the ambient environment and to contain several orders of magnitude higher concentrations than hosts. While the majority of reports mention inorganic toxin accumulation in parasites, studies concerning effects of organic pollution are infrequent and little is known about the potential of parasites to bio-accumulate microcystins. METHODS: The parasite-host system of tapeworm Khawia sinensis and common carp (Cyprinus carpio) was used to address this issue. Both the tapeworms and livers were dissected from experimental carps orally exposed to cyanobacterial biomass for 20 days. The total dose of microcystins amounted to 27 mg/kg of feed, i.e., 0.4 mg/kg of fish mass a day. Microcystin concentrations in tapeworms and carp liver tissues were measured using the LC-MS/MS method. RESULTS: Considering the three measured microcystin variants LR, YR and RR, only MC-RR was detected and its concentrations in tapeworms and carp liver tissue amounted to 5.78±3.78 ng/g and 2.11±0.74 ng/g fresh weight (p<0.05), respectively. CONCLUSIONS: Here we show accumulation of microcystin MC-RR in the tapeworm Khawia sinensis, a parasite of common carp (Cyprinus carpio). As this is the first report addressing this issue, further studies will be necessary to examine this specific parasite-host system.

Determination of the electrophoretic pattern of somatic and excretory-secretory proteins of Ligula intestinalis parasite in spirlin (Alburnoides bipunctatus).

Ligula intestinalis parasite is a cestodes that causes remarkable damages to fish. It is also of prime importance in economic and hygienic aspects. SDS-PAGE and western blotting are the methods that can be used to determine the electerophoretic pattern of somatic and excretory-secretory proteins of parasites. In this study, after obtaining the plerocercoidal stage of this parasite from the spirlin (Alburnoides bipunctatus), its somatic proteins were prepared using ultrasonicae, and excretory-secretory proteins were prepared using the PBS solution. After protein assay, which included using the Bradford method and then SDS-PAGE on these two antigenic solutions, 5 protein bands of 26, 33, 38, 58, 70kDa in somatic antigens, and 7 bands of 25, 28, 33, 43, 49, 60, 70kDa in excretory-secretory antigens were observed. After western blotting on both antigens and adding the primary antibody (the sera of infected fish) and then the secondary antibody (Rabbit Anti-fish Polyclonal Antibody Conjugated from Abnova Corporation) no band was seen in excretory-secretory antigen. And only in the 55kDa band of somatic antigen, a positive response, in comparison of fish positive serum was observed.

Perch and its parasites as heavy metal biomonitors in a freshwater environment: the case study of the Ruzín water reservoir, Slovakia.

Heavy metal concentrations were determined in 43 perches (Perca fluviatilis) and in two of its most common parasites, the acanthocephalan Acanthocephalus lucii and the cestode Proteocephalus percae, collected in the period 2009-2010 from Ruzín, a seriously polluted water reservoir in Slovakia. Samples of muscle, liver, kidney, brain, male and female reproductive organs and adipose tissue of fish and both parasites were analyzed for As, Cd, Cr, Cu, Hg, Mn, Ni, Pb and Zn, by ICP-MS. Mean concentrations of individual heavy metals in all fish samples decreased in the order zinc > copper > manganese > mercury > arsenic > chromium > cadmium > nickel > lead. Zinc was found to be the dominant element and its antagonistic interaction with copper was confirmed. The kidney was a key target organ receiving the highest mean concentrations of all analyzed metals, but some metals showed specific affinity for particular tissues. In terms of human health, concentration of Hg in fish muscle, which exceeded more than two-times its maximum level admitted in foodstuffs in European countries, is of great importance and should be taken into account. Bioaccumulation factors (C([parasite])/C([fish tissue])) calculated for all elements indicated much higher detection skills of A. lucii and P. percae parasites than fish organs and hence, present results allow proposing both parasite models as useful tools to monitor aquatic environmental quality. Acanthocephalans, however, seem to be superior for heavy metal monitoring, also demonstrated under experimental conditions. Present results also indicate the decreasing heavy metal burden of the reservoir and its gradual recovery in the course of time.

Characterization of S3Pvac anti-cysticercosis vaccine components: implications for the development of an anti-cestodiasis vaccine.

BACKGROUND: Cysticercosis and hydatidosis seriously affect human health and are responsible for considerable economic loss in animal husbandry in non-developed and developed countries. S3Pvac and EG95 are the only field trial-tested vaccine candidates against cysticercosis and hydatidosis, respectively. S3Pvac is composed of three peptides (KETc1, GK1 and KETc12), originally identified in a Taenia crassiceps cDNA library. S3Pvac synthetically and recombinantly expressed is effective against experimentally and naturally acquired cysticercosis. METHODOLOGY/PRINCIPAL FINDINGS: In this study, the homologous sequences of two of the S3Pvac peptides, GK1 and KETc1, were identified and further characterized in Taenia crassiceps WFU, Taenia solium, Taenia saginata, Echinococcus granulosus and Echinococcus multilocularis. Comparisons of the nucleotide and amino acid sequences coding for KETc1 and GK1 revealed significant homologies in these species. The predicted secondary structure of GK1 is almost identical between the species, while some differences were observed in the C terminal region of KETc1 according to 3D modeling. A KETc1 variant with a deletion of three C-terminal amino acids protected to the same extent against experimental murine cysticercosis as the entire peptide. On the contrary, immunization with the truncated GK1 failed to induce protection. Immunolocalization studies revealed the non stage-specificity of the two S3Pvac epitopes and their persistence in the larval tegument of all species and in Taenia adult tapeworms. CONCLUSIONS/SIGNIFICANCE: These results indicate that GK1 and KETc1 may be considered candidates to be included in the formulation of a multivalent and multistage vaccine against these cestodiases because of their enhancing effects on other available vaccine candidates.

Obtenção de proteína recombinante baseada em antígenos do líquido vesicular de Taenia crassiceps: aplicação no imunodiagnóstico da neurocisticercose / Recombinant protein obtainment based on antigens from Taenia crassiceps vesicular fluid: application on immunodiagnostics of neurocisticercosis

A neurocisticercose (NC) é uma doença provocada por larvas de Taenia solium (Tso) no sistema nervoso central. Seu diagnóstico fundamenta-se em critérios clínicos, epidemiológicos e laboratoriais. A utilização de antígenos parasitários no imunodiagnóstico apresenta desvantagens como: necessidade de animais, ausência de homogeneidade entre lotes, baixo rendimento, e contaminação com proteínas suínas. Assim, os antígenos recombinantes podem otimizar o imunodiagnóstico da NC, pois são reagentes simples e reprodutíveis, sem requerer animais. Este estudo teve como objetivo a obtenção, caracterização e análise da reatividade de proteína recombinante baseada em antígenos de líquido vesicular de Taenia crassiceps (Tcra). Assim, o cDNA foi obtido por amplificação a partir de RNAm de cisticercos de Tcra. A proteína recombinante Tc14 foi produzida em Escherichia coli (DE3) BL21 utilizando-se o vetor de expressão pET-22b e purificada por cromatografia de afinidade. A caracterização antigênica deu-se por Imunoblot (IB) utilizando anticorpos monoclonais (AcMo). Houve reatividade com todos os AcMo utilizados (AcMo anti-antígeno de excreção/secreção de Tcra, AcMo anti-líquido vesicular de Tcra, AcMo antilíquido vesicular de Tso e AcMo anti-antígeno total de Tso), exceto com o AcMo anti-antígeno de escólex de Tso. Utilizando-se 22 amostras de soro e 19 de líquor (LCR) de pacientes com NC, 48 soros e 28 LCR do grupo controle negativo (GCN) e 17 soros de hidatidose do grupo outras parasitoses (OP) em Imunoblot foi observada reatividade na região de 14kDa, correspondente a Tc14, em todas as amostras NC, mas não nos GCN e OP. Em ELISA com Tc14 obteve-se sensibilidade (S) e especificidade (E) de 100% com LCR (29 amostras de NC e 35 do GCN) e S de 95,1% e E de 100% com soro (41 amostras de NC, 52 do GCN). Dentre 51 soros de OP, mostraram-se reagentes um de hidatidose e outro de estrongiloidíase. A análise comparativa entre diferentes antígenos e testes sorológicos apresentou índice...

The neurocisticercosis (NC) disease is caused by the presence of Taenia solium (Tso) larvae in the central nervous system. Its diagnosis is based on clinical criteria, epidemiological studies and laboratorial exams. Nevertheless, the use of parasite antigenic extracts into the immunodiagnosis presents some disadvantages: it requires animals, lacks of homogeneity between lots, low yield and may become contaminated with swine proteins. Consequently, the utilization of recombinant antigens could optimize the immunodiagnostic of NC, as they are simple and reproducible reagents that do not require animals. This study aimed the capture, characterization and reactivity analysis of the recombinant protein based on antigens of the vesicular fluid of Taenia crassiceps (Tcra). In order to do so, the cDNA was obtained through the amplification deriving from RNAm of cysticerci of Tcra. The recombinant protein Tc14 was produced in Escherichia coli (DE3) BL21 using the expression vector pET-22b and purified by affinity chromatography (nickel resin). The antigenic characterization was performed by immunoblotting (IB) using monoclonal antibodies (MoAb). The recombinant protein presented reactivity with all the MoAb used (Anti-secretion/excretion antigens from Tcra MoAb, anti-vesicular fluid from Tcra MoAb, anti-vesicular fluid from Tso MoAb and anti- total antigen from Tso MoAb), except with the anti-antigen from Tso scolex MoAb. The immunoblot was performed using 22 serum samples and 19 cerebrospinal fluid (CSF) from patients with NC, 48 serum and 28 CSF from the negative control group (GCN) and 17 hydatidosis serum from other parasitosis' group (OP). It showed reactivity in the 14kDa region, correlated to Tc14, in all NC samples, but not presented on GCN and OP. In ELISA with Tc14, the sensibility (S) and specificity (E) of 100% was obtained with CSF (29 NC samples and 35 GCN samples) and 95.1% of S and 100% of E with serum (41 NC samples, 52 GCN samples)...

Heavy metals in the nase, Chondrostoma nasus (L. 1758), and its intestinal parasite Caryophyllaeus laticeps (Pallas 1781) from Austrian rivers: bioindicative aspects.

Tissue samples from 121 adult specimens of the predominantly herbivorous fish species nase, Chondrostoma nasus (L. 1758), from five river sites in Austria were analyzed for their metal content. Sediments and water samples of the sites show different levels of metal load, with only one site considered being polluted with metals. The concentrations of cadmium, copper, lead, and zinc in the tissue of the gills, muscle, intestine, and liver of the fish were determined by inductively coupled plasma-optical emission spectrometry (ICP-OES). As in one of the unpolluted and the polluted river site, a well-established population of the tapeworm Caryophyllaeus laticeps (Pallas, 1781) occurred in the intestine of the fish; pooled samples of this worm were analysed as well. Both the nase and C. laticeps show bioindicative ability for metal pollution in rivers. The results allow a more differentiated characterization of the rivers than the sediment analyses. Cadmium is found mainly in the liver, with maximum concentrations of 1.57 microg/g dry weight from unpolluted sites and 5.58 microg/g from the polluted site. The highest concentrations of copper are also found in the liver, with values between 25 and 333 microg/g. A significantly elevated concentration of Cu in the intestine from an "unpolluted" site (mean: 24.06 microg/g) indicates an acute pollution in this area at the time of sampling. Lead was found mainly in the intestine and liver in concentrations between 0.09 and 4.05 microg/g and 0.26 and 1.94 microg/g, respectively. In the samples from the polluted site, it also could be detected in the gills (mean: 1.38 microg/g). The parasite C. laticeps shows different capacities for metal accumulation: Although the concentrations of Cu were significantly lower compared to the values of the fish liver, cadmium was detected in concentrations up to 5.1 times higher. Lead and zinc concentrations were found to be up to 9.7 and 3.0 times higher in the tapeworm compared to the fish liver, respectively.

Heavy metal accumulation in small terrestrial rodents infected by cestodes or nematodes.

The aim of the present study was to assess whether there is a difference in accumulation of heavy metal ions (Cd, Cr, Cu, Mn, Ni, Pb and Zn) in hosts (small mammals) infected by cestode parasites when compared to those without cestode infection. The abundance of gastrointestinal parasites and bioaccumulation of heavy metals in host livers and kidneys were measured. Contents of heavy metals in hosts were determined by ICP OES method. The hosts with cestode infection (Paranoplocephala sp.) had lower contents of heavy metals in their livers and kidneys compared to hosts with nematode infection (Mastophorus muris). The content of Pb, Cd, Cr, Cu and Ni was higher in kidneys than in livers, in both (cestode and nematode infected) rodents while the content of Mn was higher in livers. Content of Zn was similar. The content of heavy metals in host was decreasing with the increasing abundance of cestodes (Paranoplocephalo sp.). Species-response models to particular heavy metals are presented.