Rapid Detection and Identification of Wuchereria bancrofti, Brugia malayi, B. pahangi, and Dirofilaria immitis in Mosquito Vectors and Blood Samples by High Resolution Melting Real-Time PCR

A simple, rapid, and high-throughput method for detection and identification of Wuchereria bancrofti, Brugia malayi, Brugia pahangi, and Dirofilaria immitis in mosquito vectors and blood samples was developed using a real-time PCR combined with high-resolution melting (HRM) analysis. Amplicons of the 4 filarial species were generated from 5S rRNA and spliced leader sequences by the real-time PCR and their melting temperatures were determined by the HRM method. Melting of amplicons from W. bancrofti, B. malayi, D. immitis, and B. pahangi peaked at 81.5+/-0.2degrees C, 79.0+/-0.3degrees C, 76.8+/-0.1degrees C, and 79.9+/-0.1degrees C, respectively. This assay is relatively cheap since it does not require synthesis of hybridization probes. Its sensitivity and specificity were 100%. It is a rapid and technically simple approach, and an important tool for population surveys as well as molecular xenomonitoring of parasites in vectors.

Cloning and characterization of a selenium-independent glutathione peroxidase (HC29) from adult Haemonchus contortus

The complete coding sequence of Haemonchus (H.) contortus HC29 cDNA was generated by rapid amplification of cDNA ends in combination with PCR using primers targeting the 5'- and 3'-ends of the partial mRNA sequence. The cloned HC29 cDNA was shown to be 1,113 bp in size with an open reading frame of 507 bp, encoding a protein of 168 amino acid with a calculated molecular mass of 18.9 kDa. Amino acid sequence analysis revealed that the cloned HC29 cDNA contained the conserved catalytic triad and dimer interface of selenium-independent glutathione peroxidase (GPX). Alignment of the predicted amino acid sequences demonstrated that the protein shared 44.7~80.4% similarity with GPX homologues in the thioredoxin-like family. Phylogenetic analysis revealed close evolutionary proximity of the GPX sequence to the counterpart sequences. These results suggest that HC29 cDNA is a GPX, a member of the thioredoxin-like family. Alignment of the nucleic acid and amino acid sequences of HC29 with those of the reported selenium-independent GPX of H. contortus showed that HC29 contained different types of spliced leader sequences as well as dimer interface sites, although the active sites of both were identical. Enzymatic analysis of recombinant prokaryotic HC29 protein showed activity for the hydrolysis of H2O2. These findings indicate that HC29 is a selenium-independent GPX of H. contortus.

Non-coding RNAs in schistosomes: an unexplored world

Non-coding RNAs (ncRNAs) were recently given much higher attention due to technical advances in sequencing which expanded the characterization of transcriptomes in different organisms. ncRNAs have different lengths (22 nt to >1, 000 nt) and mechanisms of action that essentially comprise a sophisticated gene expression regulation network. Recent publication of schistosome genomes and transcriptomes has increased the description and characterization of a large number of parasite genes. Here we review the number of predicted genes and the coverage of genomic bases in face of the public ESTs dataset available, including a critical appraisal of the evidence and characterization of ncRNAs in schistosomes. We show expression data for ncRNAs in Schistosoma mansoni. We analyze three different microarray experiment datasets: (1) adult worms' large-scale expression measurements; (2) differentially expressed S. mansoni genes regulated by a human cytokine (TNF-α) in a parasite culture; and (3) a stage-specific expression of ncRNAs. All these data point to ncRNAs involved in different biological processes and physiological responses that suggest functionality of these new players in the parasite's biology. Exploring this world is a challenge for the scientists under a new molecular perspective of host-parasite interactions and parasite development.

RNAs não codificadores (ncRNAs) têm sido recentemente objeto de atenção muito maior devido aos avanços técnicos no sequenciamento que expandiram a caracterização dos transcritomas em diferentes organismos. ncRNAs possuem diferentes comprimentos (22 nt a >1.000 nt) e mecanismos de ação que essencialmente compreendem uma sofisticada rede de regulação de expressão gênica. A publicação recente dos genomas e transcritomas dos esquistossomos aumentou a descrição e caracterização de um grande número de genes do parasita. Aqui nós revisamos o número de genes preditos e a cobertura das bases do genoma em face dos ESTs públicos disponíveis, incluindo uma avaliação crítica da evidência e caracterização de ncRNAs em esquistossomos. Nós mostramos dados de expressão de ncRNAs em Schistosoma mansoni. Nós analisamos três conjuntos diferentes de dados de experimentos com microarranjos: (1) medidas de expressão em larga escala de vermes adultos; (2) genes diferencialmente expressos de S. mansoni regulados por uma citocina humana (TNF-α) no parasita em cultura; e (3) expressão estágio-especifica de ncRNAs. Todos estes dados apontam para ncRNAs envolvidos em diferentes processos biológicos e respostas fisiológicas que sugerem funcionalidade destes novos personagens na biologia do parasita. Explorar este mundo é um desafio para os cientistas sob uma nova perspectiva molecular da interação parasita-hospedeiro e do desenvolvimento do parasita.

A genome-wide RNAi screen identifies genes regulating the formation of P bodies in C. elegans and their functions in NMD and RNAi

Cytoplasmic processing bodies, termed P bodies, are involved in diverse post-transcriptional processes including mRNA decay, nonsense-mediated RNA decay (NMD), RNAi, miRNA-mediated translational repression and storage of translationally silenced mRNAs. Regulation of the formation of P bodies in the context of multicellular organisms is poorly understood. Here we describe a systematic RNAi screen in C. elegans that identified 224 genes with diverse cellular functions whose inactivations result in a dramatic increase in the number of P bodies. 83 of these genes form a complex functional interaction network regulating NMD. We demonstrate that NMD interfaces with many cellular processes including translation, ubiquitin-mediated protein degradation, intracellular trafficking and cytoskeleton structure.We also uncover an extensive link between translation and RNAi, with different steps in protein synthesis appearing to have distinct effects on RNAi efficiency. Moreover, the intracellular vesicular trafficking network plays an important role in the regulation of RNAi. A subset of genes enhancing P body formation also regulate the formation of stress granules in C. elegans. Our study offers insights into the cellular mechanisms that regulate the formation of P bodies and also provides a framework for system-level understanding of NMD and RNAi in the context of the development of multicellular organisms.

Characterization of HC58cDNA, a putative cysteine protease from the parasite Haemonchus contortus

Because of the complexity of the cathepsin B-like (CBL) family, an information on the biological and biochemical characteristics of individual CBL genes is lacking. In this study, we investigated the degradative effects of the recombinant HC58 protein isolated from Haemonchus contortus parasites on protein substrates over a broad pH range in vitro. This protein, which hydrolyzed the synthetic peptide substrates Z-FR-AMC and Z-RR-AMC, had characteristics of the cysteine protease class of proteins. In the acidic pH range, the isolated protein actively degraded hemoglobin (Hb), the heavy chain of goat immunoglobulin G, and azocasein. By contrast, it degraded fibrinogen in the alkaline pH range. These activities were strongly inhibited in the presence of the cysteine protease inhibitor E-64. While the protein digested Hb, it did not induce the agglutination of erythrocytes from its natural host. These results suggest that the HC58 protein may play a role in the nutrition of this parasite.

Enterobius vermicularis: ancient DNA from north and south American human coprolites

A molecular paleoparasitological diagnostic approach was developed for Enterobius vermicularis. Ancient DNA was extracted from 27 coprolites from archaeological sites in Chile and USA. Enzymatic amplification of human mtDNA sequences confirmed the human origin. We designed primers specific to the E. vermicularis 5S ribosomal RNA spacer region and they allowed reproducible polymerase chain reaction identification of ancient material. We suggested that the paleoparasitological microscopic identification could accompany molecular diagnosis, which also opens the possibility of sequence analysis to understand parasite-host evolution

Cloning of a pore-forming subunit of ATP-sensitive potassium channel from Clonorchis sinensis

A complete cDNA sequence encoding a pore-forming subunit (Kir6.2) of ATP-senstive potassium channel in the adult worm, Clonorchis sinensis, termed CsKir6.2, was isolated from an adult cDNA library. The cDNA contained a single open-reading frame of 333 amino acids, which has a structural motif (a GFG-motif) of the putative pore-forming loop of the Kir6.2. Peculiarly, the CsKir6.2 shows a lack-sequence structure, which deleted 57 amino acids were deleted from its N-terminus. The predicted amino acid sequence revealed a highly conserved sequence as other known other Kir6.2 subunits. The mRNA was weekly expressed in the adult worm.

Partial molecular characterization of Sm8, a tegumental antigen of Schistosoma mansoni

Sm8 is a major tegumental antigen of Schistosoma mansoni. The partial cDNA was isolated and analyzed. Sequence analysis revealed transmembrane compatible hydrophobic domains and a putative leucine zipper pattern. The mRNA and the protein are predominantly expressed in adult worms

Studies on the development of DNA vaccine against Cysticercus cellulosae infection and its efficacy.

DNA vaccine against Cysticercus cellulosae infection was developed and its efficacy was tested. A pair of primers specific to antigen B gene of C. cellulosae was designed which amplified the gene successfully with RT-PCR. The gene was ligated to PV93 vector, and the recombinant of antigen B gene and PV93 was transformed to JM83 cells. The transformed JM83 cells were cultured in a large scale and the plasmid purified. Based on the recombinant plasmid. a DNA vaccine was developed and used to vaccinate two groups of experimental pigs. In each group, there was a routine vaccine, an enhanced vaccine and a control group. Groups 1 and 2 were challenged at 4 months and at 14 days post vaccination respectively with eggs of Taenia solium. The antibody response was also tested with ELISA. The results suggested that all animals vaccinated AgB gene DNA vaccine, no matter by routine or enhanced vaccine, their antibodies reached maximum peak 23 days post vaccination and decreased gradually. When the animals were challenged 4 months after vaccination, they had strong immunity and the parasites decrease rates were 91.2% and 93.1% respectively. When pigs vaccinated with AgB gene DNA vaccine were challenged 14 days post vaccination with 18,000 eggs/pig. The animals showed strong immunity and the parasite decrease rates were 99.5% and 84.9% respectively. However at that time, the antibodies did not reach the peak. While in the control group, the number of C. cellulosae was as many as 2,500. It was concluded that the pigs vaccinated with DNA vaccine had strong immunity against infection of eggs of T. solium.

Histochemical alterations of infective third-stage hookworm larvae (L3) in vaccinated mice.

To study the histochemical alterations of hookworm L3 administered in a challenge dose to mice vaccinated previously with the larvae. Male Kunming strain mice vaccinated subcutaneously with 500 living Ancylostoma caninum L3 once every 2 weeks for a total of three immunizations before a final challenge with 500 L3 one week after the final immunization. The abdominal skin with underlying subcutaneous tissue and muscle were removed from the site of percutaneous challenge entry (from 2-3 mice), and fixed in absolute alcohol, cold acetone and 10% neutralized formalin. The tissue sections containing the L3 from the challenge dose were then stained histochemically of glycogen, RNA, DNA alkaline protein, acid mucopolysaccharide, collagen, reticulin, alkaline phosphatase (AKP) and adenosine triphosphatase (ATPase). Skin samples from non-immunized mice that were also subcutaneously inoculated with the L3 served as negative control. The L3 identified in cutaneous sections from vaccinated mice at 6-72 hours post-challenge exhibited reductions in parasite glycogen, alkaline protein, RNA and DNA, as well as reductions in acid mucopolysaccharide, collagen and reticulin contents in the parasite cuticle. There were also reduced enzyme AKP and ATPase activities. In contrast L3, identified in sections from non-immunized mice exhibited a normal histochemical appearance, as did some L3 who survived in vaccinated mice at 7-14 days post-challenge. Vaccination results in hookworm L3 damage which is manifested by reduced histochemical staining for the challenge inoculum of parasites. There is also reduced hydrolytic enzyme activity. The observed changes could reflect either host-mediated parasite structural damage and disintegration or possibly anti-metabolic properties of the host immune response.

Cloning and characterization of a Schistosoma mansoni cDNA clone with a specific antigenic expression during development in a vertebrate host

Approximately 2.0 x 10 cDNA clones of an Schistosoma mansoni gt11 cDNA library were screened in duplicate with serum from infected mice corresponding to distinct phases of infection. A cDNA clone (7/1) was isolated and recognized only by seven week serum. The clone was subcloned in pGEX-2T and Western-blot studies showed a specific antigenic expression confirming that only serum from the chronic phase is capable of recognizing this antigen. Dot-hybridization with RNA from different developmental phases of the parasite showed that the corresponding 7/1 RNA is expressed in all phases of parasite development in vertebrate hosts.

Cytochemistry of ribonucleic acid [RNA] in murine liver cells infected with schistosoma mansoni

The changes in the concentration of ribonucleic acid [RNA] were studied in liver cells of mouse after infestation with the cercariae of Schistosoma mansoni. It was found, that there is a fluctuation in the intensity of basophilia in parenchymal cells depending on the interval of schistosomal infection. Using the methyl green-pyronin stain, it was found that RNA is localized in the nucleolus and cytoplasm of both normal and infected hepatic cells of mouse. Pronnounced changes in the concentration of RNA were observed in hepatic cells after 50, 75, and 100 days from the beginning of schistosomal infestation, These changes include a decrease in the intensity of basophilia in 50 days post infection and its increase in 75 and 100 days of infection respectively