Detection of Opisthorchis viverrini antigens in stools using specific monoclonal antibody.

Detection of Opisthorchis viverrini antigens in stools using specific monoclonal antibody. International Journal for Parasitology 22: 527-531. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for detecting Opisthorchis viverrini antigen in faecal extracts of four groups of individuals. These were 24 patients with O. viverrini infection only (group 1), 31 patients with O. viverrini and other parasitic infections (group 2), 141 patients with other parasitic infections (group 3) and 21 normal, parasite-free individuals (group 4). The first antibody used in the ELISA was polyclonal immunoglobulin G prepared from the serum of a rabbit previously immunized with crude extract of O. viverrini. The second antibody was monoclonal antibody specific to an antigen located in the worm tegument and muscular tissue. Sensitivity of the assay was 31% while specificity was 100%. Considerations for improving the sensitivity are discussed.     

Affinities between Asian non-human Schistosoma species,the S. indicum group,and the African human schistosomes

Schistosoma species have traditionally been arranged in groups based on egg morphology, geographical origins, and the genus or family of snail intermediate host. One of these groups is the 'S. indicum group' comprising species from Asia that use pulmonate snails as intermediate hosts. DNA sequences were obtained from the four members of this group (S. indicum, S. spindale, S. nasale and S. incognitum) to provide information concerning their phylogenetic relationships with other Asian and African species and species groups. The sequences came from the second internal transcribed spacer (ITS2) of the ribosomal gene repeat, part of the 28S ribosomal RNA gene (28S), and part of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene. Tree analyses using both distance and parsimony methods showed the S. indicum group not to be monophyletic. Schistosoma indicum, S. spindale and S. nasale were clustered among African schistosomes, while S. incognitum was placed as sister to the African species (using ITS2 and 28S nucleotide sequences and CO1 amino acid sequences), or as sister to all other species of Schistosoma (CO1 nucleotide sequences). Based on the present molecular data, a scenario for the evolution of the S. indicum group is discussed.     

Schistosoma mekongi and Schistosoma japonicum: Differences in the distribution of eggs in the viscera of mice

The difference in the distribution of Schistosoma eggs in the viscera has not been clearly elucidated in the two closely related species Schistosoma japonicum and Schistosoma mekongi. In this study, we quantitatively compared the distribution of eggs in mice infected with the two species. In S. mekongi-infected mice, 56.6% to 69.4% of total eggs were found in the distal small intestine 9 to 15 weeks after infection, while in S. japonicum-infected mice, 48.8% to 71.8% of eggs were found in the proximal small intestine during the same period. There were significantly more eggs in the liver in mice infected with S. japonicum than in those infected with S. mekongi. The number of adult worms recovered did not differ between the two species during the study period. The total number of eggs laid in the tissues also did not differ between the two species at 12 to 15 weeks postinfection, but in the earlier period the total number of eggs was significantly fewer in S. mekongi-infected than in S. japonicum-infected mice, suggesting the delayed maturation of the former compared with the latter. These results clearly show that S. japonicum and S. mekongi exhibit different oviposition behavior in their hosts.     

Schistosoma mekongi: a prominent neutrophil chemotactic activity of egg antigen with reference to that of Schistosoma japonicum

Schistosoma mekongi causes granulomatous lesions around eggs deposited in the liver with neutrophil-rich inflammatory reactions in the early stage of the egg laying. To define the aspects of the typical pathogenesis of S. mekongi infection, we determined the difference between soluble egg antigen (SEA) from S. mekongi and S. japonicum with a focus on chemotactic factors for neutrophils or eosinophils. Mean volume and protein amount of S. mekongi eggs was 71 and 58% of those of Schistosoma japonicum eggs, respectively. Neutrophil chemotactic activity of S. mekongi SEA was about two times higher than that of S. japonicum. In contrast, eosinophil chemotactic activity of S. mekongi SEA was about half of that of S. japonicum SEA. Molecular analysis revealed that S. mekongi SEA contains higher molecular-weight components with a lower level of glycosylation, and this is likely to be related to the intense neutrophil chemotactic activity in comparison with S. japonicum SEA. The prominent chemotactic reactivity for neutrophils is likely to be involved in the typical pathogenesis of mekongi schistosomiasis.     

Fluorescence resonance energy transfer analysis of subunit stoichiometry of the epithelial Na+ channel

Activity of the epithelial Na(+) channel (ENaC) is rate-limiting for Na(+) (re)absorption across electrically tight epithelia. ENaC is a heteromeric channel comprised of three subunits, alpha, beta, and gamma, with each subunit contributing to the functional channel pore. The subunit stoichiometry of ENaC remains uncertain with electrophysiology and biochemical experiments supporting both a tetramer with a 2alpha:1beta:1gamma stoichiometry and a higher ordered channel with a 3alpha:3beta:3gamma stoichiometry. Here we used an independent biophysical approach based upon fluorescence resonance energy transfer (FRET) between differentially fluorophore-tagged ENaC subunits to determine the subunit composition of mouse ENaC functionally reconstituted in Chinese hamster ovary and COS-7 cells. We found that when all three subunits were co-expressed, ENaC contained at least two of each type of subunit. Findings showing that ENaC subunits interact with similar subunits in immunoprecipitation studies are consistent with these FRET results. Upon native polyacrylamide gel electrophoresis, moreover, oligomerized ENaC runs predominantly as a single species with a molecular mass of >600 kDa. Because single ENaC subunits have a molecular mass of approximately 90 kDa, these results also agree with the FRET results. The current results as a whole, thus, are most consistent with a higher ordered channel possibly with a 3alpha:3beta:3gamma stoichiometry.     

Differential diagnosis of schistosomiasis mekongi and trichinellosis in human.

An indirect (plate) ELISA and, a more convenient version, a dot-blot (membrane) ELISA have been developed using haemocyanin of a mollusk, Megathura crenulata, i.e. keyhole limpet haemocyanin (KLH) and purified, specific antigen of Trichinella spiralis (APTsAg) obtained from a monoclonal antibody-affinity column chromatography, for differential diagnosis of schistosomiasis mekongi and trichinellosis. Serum samples of patients with parasitologically confirmed trichinellosis were reactive to both antigens in both versions of ELISA while sera of patients with schistosomiasis mekongi were positive only to the KLH. Both ELISA were negative when used to test sera of normal controls and patients with gnathostomiasis, paragonimiasis and opisthorchiasis.     

Genome 3'-end repair in dengue virus type 2

Genomes of RNA viruses encounter a continual threat from host cellular ribonucleases. Therefore, viruses have evolved mechanisms to protect the integrity of their genomes. To study the mechanism of 3′-end repair in dengue virus-2 in mammalian cells, a series of 3′-end deletions in the genome were evaluated for virus replication by detection of viral antigen NS1 and by sequence analysis. Limited deletions did not cause any delay in the detection of NS1 within 5 d. However, deletions of 7–10 nucleotides caused a delay of 9 d in the detection of NS1. Sequence analysis of RNAs from recovered viruses showed that at early times, virus progenies evolved through RNA molecules of heterogeneous lengths and nucleotide sequences at the 3′ end, suggesting a possible role for terminal nucleotidyl transferase activity of the viral polymerase (NS5). However, this diversity gradually diminished and consensus sequences emerged. Template activities of 3′-end mutants in the synthesis of negative-strand RNA in vitro by purified NS5 correlate well with the abilities of mutant RNAs to repair and produce virus progenies. Using the Mfold program for RNA structure prediction, we show that if the 3′ stem–loop (3′ SL) structure was abrogated by mutations, viruses eventually restored the 3′ SL structure. Taken together, these results favor a two-step repair process: non-template-based nucleotide addition followed by evolutionary selection of 3′-end sequences based on the best-fit RNA structure that can support viral replication.     

Biochemical Aspects of Parasitism by the Angiosperm Parasites: Changes in Nucleic Acid Content of Infected Hosts

The acid-insoluble phosphate in the tissues of three species of Cuscuta and of Orobanche cernua and in the infected hosts has been fractionated into phospholipids, RNA, DNA and phosphoprotein. Infection by parasites was attended, in general, by a decrease in RNA and an increase in DNA.