Evaluating integrated urban water systems alternatives for Brisbane, Australia.

The Brazil Development Study investigates the feasibility of alternative approaches to providing sustainable water services to a 226 ha mixed residential and industrial greenfield development within the city of Brisbane, Australia. The alternatives include techniques such a the use of rainwater tanks, water use efficiency, a local wastewater treatment plant for recycling of reclaimed water and composting toilets amongst others. This paper evaluates a series of urban development scenarios against the objectives of the study. The insights gained into the drivers for cost and environmental impact for this particular site are discussed as well as a number of issues of concern and challenges to Council and the community.

Severe reactions to filarial chemotherapy and release of Wolbachia endosymbionts into blood.

Wolbachia bacteria seem to have evolved as essential endosymbionts of their filarial nematode hosts. Studies in mice have suggested that these bacteria are associated with systemic inflammatory reactions to filarial chemotherapy. We took blood samples from 15 Indonesian patients before and after treatment with diethylcarbamazine for Brugia malayi infection, and recorded the severity of any post-treatment inflammatory reactions. Blood from all three patients with severe adverse reactions and from one of six with moderate reactions was positive for Wolbachia DNA 4-48 h after diethylcarbamazine treatment. We suggest that these severe inflammatory reactions are associated with the release of endosymbionts into the blood after treatment for filariasis.

Expression and secretion of a larval-specific chitinase (family 18 glycosyl hydrolase) by the infective stages of the parasitic nematode, Onchocerca volvulus.

A recently reported chitinase gene, expressed in the infective, third-stage (L3) larvae of the human filarial parasite Onchocerca volvulus, belongs to the family 18 glycosyl hydrolases and has been designated Ov-chi-1. The gene product of Ov-chi-1 is chitinolytic. Allosamidin ablates activity of the native enzyme in a dose-dependent manner but did not significantly inhibit the moulting of L3 larvae. Mono-specific antibodies were used to characterize Ov-CHI-1 as a 60-kDa protein expressed almost exclusively in L3 stages. Immunoelectron microscopy showed that Ov-CHI-1 expression is initiated in late L2 larvae and increases markedly in infective, L3 larvae. It is synthesized exclusively in the glandular esophagus and stored within discrete secretory granules. Secretion occurs through de-granulation during post-infective development, and the primary route of transport appears to be via the pseudo-coelom. An orthologue of Ov-chi-1 was detected in Caenorhabditis elegans by BLAST analysis. It is constitutively expressed at a low level and is overexpressed in dauer larvae and embryonated eggs. It is chitinolytic. We conclude that Ov-CHI-1 is a highly stage-specific enzyme that may have a role in infectivity of the parasite, aiding escape from the vector or participating in early post-infective migration and/or development. The identification of an orthologue in C. elegans opens the way for further studies into the biological function(s) of this intriguing parasite product.

Characterization of the heat-shock protein 60 chaperonin from Onchocerca volvulus.

Chaperonin 60 (cpn60) belongs to the group of ubiquitous molecular chaperones that comprise the heat shock proteins, nucleoplasmins and chaperonins. Antibodies to recombinant CPN60 from humans was used to screen a cDNA library of Onchocerca volvulus and antigen-positive clones were selected. Sequencing of the DNA inserts confirmed their identity as cpn60 transcripts. These are distinct from a cpn60 sequence recorded previously from O. volvulus (GenBank accession number Y09416) that appears to be of endobacterial origin, rather than derived from the parasite itself. The full-length sequence of the cDNA (designated Ov-cpn60) codes for a protein of 64.3kDa (598 amino acid residues) and shares significant identity with homologous gene products from Caenorhabditis elegans (72%), humans (69%), yeast (53%) and Escherichia coli (50%). The endobacterial and parasite sequences are 41% conserved. Ov-CPN60 migrates with an apparent molecular mass of 65kDa on SDS-PAGE and is present in all life-cycle stages, as determined by immunoblotting with rabbit antibodies raised against the recombinant protein. Immunogold electron microscopy identified the protein within mitochondria, as expected, but also in extra-mitochondrial sites, including inclusion bodies of the glandular oesophagus (in infective larvae), the uterine wall, cytosol of developing spermatids, and the hypodermis and cuticle. Endobacteria were also labelled, indicating cross-reactivity between CPN60 from the parasite and its intracellular symbiont. In human infections, serum antibodies to Ov-CPN60 were present in only 11% of cases from Ecuador, but in 81-89% of subjects in three separate foci from West Africa. There was no relationship between antibody levels and age, sex, or infection intensity, and no consistent association between the serological response and immune status. An evaluation of antibody specificities in individual sera revealed a mixture of parasite-specific and host crossreactive anti-CPN60 antibodies, the ratio of which varied amongst geographic areas. It is concluded that antibody responses to Ov-CPN60 are unlikely to contribute either to host protection or pathology in onchocerciasis.

DNA immunisation with Onchocerca volvulus chitinase induces partial protection against challenge infection with L3 larvae in mice.

The parasitic nematode, Onchocerca volvulus is a major cause of blindness and dermal pathology in tropical regions. A vaccine directed to infective larvae would provide a valuable control tool alongside the current methods of chemotherapy and vector control. Previously we have described the identification of a chitinase cDNA that is expressed in a stage specific manner by O. volvulus infective third stage (L3) larvae. To evaluate its host protective potential, the complete open reading frame was cloned into the eukaryotic expression plasmid pJW4303 and used to vaccinate mice by DNA immunisation with the Accell GeneGun. The survival of challenge infective larvae was monitored using implanted micropore chambers. In the first trial, mice immunised 3 times over 4 months with 1 microg O. volvulus chitinase DNA responded with modest antibody responses dominated by IgG2a and exhibited a 36% (p=0.189, NS) reduction in parasite survival compared with challenge controls. In the second trial, an increased dose of DNA (5 microg) and more frequent immunisations (5 times over 6 months) stimulated an IgG1 dominant response and a 53% reduction in parasite survival (p=0.042). Antibodies from the vaccinated mice reacted with the cuticle of post-infective L3 larvae, implying that this may be the site of immune attack following secretion of chitinase.

Thioredoxin peroxidase from Onchocerca volvulus: a major hydrogen peroxide detoxifying enzyme in filarial parasites.

Random screening of an Onchocerca volvulus third-stage (L3) cDNA library identified a highly abundant cDNA encoding a newly discovered antioxidant enzyme, thioredoxin peroxidase (TPx), a member of the peroxidoxin superfamily. This TPx cDNA (Ov-tpx-2) encodes a polypeptide of 199 amino acid residues with a calculated molecular weight of 21,890 Da. The Ov-tpx-2 cDNA represents roughly 2.5% of the total cDNAs from the L3 cDNA library. The gene was expressed in Escherichia coli and the protein product was shown to have antioxidant activity. Antiserum raised against Ov-TPX-2 recognized a native protein from extracts of both the L3 and adult-stages with a molecular weight of 22 kD. The localization and stage-specificity of Ov-TPX-2 protein was analyzed by immunocytochemistry and immunoelectron microscopy using monospecific antibodies. Expression was detected in late first-stage larvae during development in the vector and increased in intensity during differentiation to the infective L3-stage. The antigen was also detected in post-infective larvae and adult worms. In larvae, Ov-TPX-2 protein was predominantly localized to the hypodermis and cuticle, with additional sites in the hypodermal chords and multivesicular bodies. In adult worms, the primary sites of expression were the uterine epithelium and intestine, with additional labeling of the body wall and cuticle. Developing embryos and microfilariae in utero were bathed in Ov-TPX-2 protein discharged from epithelial cells. These results suggest that Ov-TPX-2 may protect the parasites from being damaged by host-generated oxidative stress and that Ov-TPX-2 protein provides the H2O2-detoxifying activity predicted but not previously identified in filarial parasites. Its highly upregulated expression in infective larvae may aid in parasite establishment following transmission to the definitive host.