RESUMO
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.
Assuntos
Cestoides/química , Doenças dos Peixes/parasitologia , Linguado/parasitologia , Larva/química , Hidrocarbonetos Policíclicos Aromáticos/análise , Animais , Bioacumulação/fisiologia , Golfo do México , Espectrometria de FluorescênciaRESUMO
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.
Assuntos
Larva/metabolismo , Estágios do Ciclo de Vida , Proteômica/métodos , Animais , Cestoides/química , Infecções por Cestoides/diagnóstico , Infecções por Cestoides/tratamento farmacológico , Proteínas de Helminto/análise , Proteínas de Helminto/fisiologia , Humanos , Mesocestoides/química , Reprodução Assexuada , Diferenciação SexualRESUMO
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)...
Assuntos
Humanos , Feminino , Camundongos , Cestoides/química , Técnicas In Vitro , Neurocisticercose , Proteínas Recombinantes/análise , Testes Imunológicos/estatística & dados numéricos , Antígeno Nuclear de Célula em Proliferação/análise , Ensaio de Imunoadsorção Enzimática , Interpretação Estatística de DadosRESUMO
Antigen B (AgB), an immunodominant component of the cestode parasite Echinococcus granulosus, presents homology to and shares apparent structural similarities with helix-rich hydrophobic ligand binding proteins (HLBPs) from other cestodes. In order to investigate the fatty acid binding properties of AgB, two of its subunit components (rAgB8/1 and rAgB8/2) were expressed in Escherichia coli and purified, and the native antigen was purified from the hydatid cyst fluid by affinity chromatography using a monoclonal antibody raised against rAgB8/1. The interaction of the purified native and recombinant proteins with the fluorescent ligands DAUDA, ANS, DACA and 16-AP was investigated. The palmitic acid derived fluorescent ligand, 16-AP, showed the greatest enhancement in fluorescence when bound to native AgB or to its recombinant subunits, and the dissociation constants for 16-AP binding were determined. Surprisingly, in contrast to HLBPs from other cestodes, interactions with other fatty acids, including palmitic acid, caused an increase in fluorescence instead of competing with 16-AP. Our results suggest that AgB might have evolved different functions in the binding of hydrophobic compounds, dependent on cestode environment.
Assuntos
Ácidos Graxos/metabolismo , Proteínas de Helminto/química , Interações Hidrofóbicas e Hidrofílicas , Lipoproteínas/química , Animais , Anticorpos Anti-Helmínticos/imunologia , Anticorpos Monoclonais/imunologia , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cestoides/química , Cestoides/metabolismo , Cromatografia de Afinidade , Líquido Cístico/metabolismo , Escherichia coli/genética , Ácidos Graxos/química , Proteínas de Helminto/imunologia , Proteínas de Helminto/metabolismo , Cinética , Ligantes , Lipoproteínas/imunologia , Lipoproteínas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Espectrometria de FluorescênciaRESUMO
A specific, precise and accurate high-performance liquid chromatographic (HPLC) analytical method has been developed for the quantitative determination of different benzimidazole (BZD) anthelmintics in parasite material (Moniezia benedeni). Mebendazole (MBZ), oxibendazole (OBZ), flubendazole (FLBZ), albendazole (ABZ) ricobendazole (RBZ), albendazole sulphone (ABZSO(2)), fenbendazole (FBZ), oxfendazole (OFZ) and fenbendazole sulphone (FBZSO(2)) were measured simultaneously in M. benedeni, a sheep and cattle cestode parasite used as a model of the biological matrix. The recovery, linearity, precision, accuracy, limits of detection and quantification of the method were determined. Drug extraction from the parasite's tissue homogenate was performed using methanol (liquid phase extraction), and after solvent evaporation, the residual material was cleaned up by solid phase extraction prior to analysis by reversed-phase HPLC. The resolution of all the BZD molecules assayed was obtained on a C(18) reversed-phase (5 microm, 250 mm x 4.6 mm) column using acetonitrile and ammonium acetate as the mobile phase and ultraviolet (UV) detection at 292 nm. Regression analyses for all the BZD compounds assayed were linear at concentrations ranging from 1.61 to 64.21 nmol/100mg protein (triplicate determinations) showing correlation coefficients greater than 0.9922. The developed method is efficient for the simultaneous determination of several benzimidazole anthelmintic molecules in parasite material and useful for the ex vivo and in vivo characterisation of the kinetics of drug uptake/diffusion in target parasites, which seems to be relevant to optimise parasite control both in human and veterinary medicine.