RESUMO
Disease surveillance in Neotropical primates (NP) is limited by the difficulties associated with anesthetizing NP for sample collection in remote settings. Our objective was to optimize a noninvasive method of oral sampling from semicaptive NP in Peru. We offered 40 NP at Taricaya Rescue Centre in Madre de Dios, Peru ropes coated in various attractants and measured variables (acceptance of the rope, chewing time, and volume of fluid eluted from ropes) that may affect sample acquisition and quality. We preserved samples by direct freezing in liquid nitrogen or by storing samples in RNA stabilization reagent at room temperature. Sample integrity was measured by testing for mammalian cytochrome b with the use of conventional PCR. The NP successfully chewed on a rope in 82% (125/152) of trials. Overall sample integrity was high, with 96% (44/46) of samples (both directly frozen and stored in stabilization reagent) testing positive for cytochrome b. The number of times that an individual NP was exposed to the rope procedure and NP age were associated with higher acceptance rates and the NP successfully chewing on the rope. We conclude that ropes serve as a feasible noninvasive method of obtaining oral samples from NP at rescue centers and could be used in future studies to evaluate population genetics and for pathogen surveillance for population health monitoring.
Assuntos
Haplorrinos , Saliva , Manejo de Espécimes/veterinária , Envelhecimento , Animais , Comportamento Animal , Feminino , Masculino , BocaRESUMO
BACKGROUND: It is believed that schistosomes evade complement-mediated killing by expressing regulatory proteins on their surface. Recently, six homologues of human CD59, an important inhibitor of the complement system membrane attack complex, were identified in the schistosome genome. Therefore, it is important to investigate whether these molecules could act as CD59-like complement inhibitors in schistosomes as part of an immune evasion strategy. METHODOLOGY/PRINCIPAL FINDINGS: Herein, we describe the molecular characterization of seven putative SmCD59-like genes and attempt to address the putative biological function of two isoforms. Superimposition analysis of the 3D structure of hCD59 and schistosome sequences revealed that they contain the three-fingered protein domain (TFPD). However, the conserved amino acid residues involved in complement recognition in mammals could not be identified. Real-time RT-PCR and Western blot analysis determined that most of these genes are up-regulated in the transition from free-living cercaria to adult worm stage. Immunolocalization experiments and tegument preparations confirm that at least some of the SmCD59-like proteins are surface-localized; however, significant expression was also detected in internal tissues of adult worms. Finally, the involvement of two SmCD59 proteins in complement inhibition was evaluated by three different approaches: (i) a hemolytic assay using recombinant soluble forms expressed in Pichia pastoris and E. coli; (ii) complement-resistance of CHO cells expressing the respective membrane-anchored proteins; and (iii) the complement killing of schistosomula after gene suppression by RNAi. Our data indicated that these proteins are not involved in the regulation of complement activation. CONCLUSIONS: Our results suggest that this group of proteins belongs to the TFPD superfamily. Their expression is associated to intra-host stages, present in the tegument surface, and also in intra-parasite tissues. Three distinct approaches using SmCD59 proteins to inhibit complement strongly suggested that these proteins are not complement inhibitors and their function in schistosomes remains to be determined.
Assuntos
Proteínas do Sistema Complemento/metabolismo , Proteínas de Helminto/química , Proteínas de Helminto/imunologia , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Schistosoma mansoni/química , Schistosoma mansoni/imunologia , Animais , Cricetinae , Feminino , Humanos , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/imunologia , Modelos Moleculares , Conformação ProteicaRESUMO
BACKGROUND: Schistosomiasis mansoni is a debilitating and sometimes fatal disease. Accurate diagnosis plays a key role in patient management and infection control. However, currently available parasitological methods are laborious and lack sensitivity. The selection of target antigen candidates has turned out to be a promising tool for the development of more sensitive diagnostic methods. In our previous investigations, the use of crude antigens led to false-positive results. Recently, focus has been given to highly purified Schistosoma mansoni antigens, especially to circulating antigens. METHOD: Thus, our main goal was to test different types of circulating cathodic antigen glycoprotein (CCA), as "crude antigen," the protein chain of recombinant CCA and two individual peptides. These schistosome proteins/peptides were tested in a new diagnostic method employing immunomagnetic separation based on the improvement of antigen-antibody binding. PRINCIPAL FINDINGS: Use of recombinant CCA as a diagnostic antigen allowed us to develop a diagnostic assay with high sensitivity and specificity with no false-negative results. Interestingly, the "crude antigen" worked as a good marker for control of cure after praziquantel treatment. CONCLUSIONS/SIGNIFICANCE: Our new diagnostic method was superior to enzyme-linked immunosorbent assay in diagnosing low endemicity patients.