Filarial nematodes possess glutathione transferases (GSTs), ubiquitous enzymes with the potential to detoxify xenobiotic and endogenous substrates, and modulate the host immune system, which may aid worm infection establishment, maintenance and survival in the host. Here we have identified and characterized a σ class glycosylated GST (OoGST1), from the cattle-infective filarial nematode Onchocerca ochengi, which is homologous (99% amino acid identity) with an immunodominant GST and potential vaccine candidate from the human parasite, O. volvulus, (OvGST1b). Onchocerca ochengi native GSTs were purified using a two-step affinity chromatography approach, resolved by 2D and 1D SDS-PAGE and subjected to enzymic deglycosylation revealing the existence of at least four glycoforms. A combination of lectin-blotting and mass spectrometry (MS) analyses of the released N-glycans indicated that OoGST1 contained mainly oligomannose Man5GlcNAc2 structure, but also hybrid- and larger oligommanose-type glycans in a lower proportion. Furthermore, purified OoGST1 showed prostaglandin synthase activity as confirmed by Liquid Chromatography (LC)/MS following a coupled-enzyme assay. This is only the second reported and characterized glycosylated GST and our study highlights its potential role in host-parasite interactions and use in the study of human onchocerciasis.

The presence of retinol-binding protein (RBP) activity in Onchocerca cervicalis adult worms and interaction with ivermectin has been studied using high pressure size exclusion chromatography (HPSEC). Four distinct peaks of [3H]-retinol incorporation were obtained corresponding to approximate molecular weights of 150, 67, 19·7 and 4–6 kDa, the 2 smaller Mr peaks accounting for most of the binding activity. Competition for binding using non-labelled retinol at 200-fold molar excess indicated that specific binding of retinol occurred only to the 19–7 kDa fraction. Competition by ivermectin also inhibited binding of [3H]-retinol to the third peak. Following incubation with [3H]-ivermectin & peaks of similar molecular weights were also detected by HPSEC in soluble adult worm homogenate, However, in this case the 150 kDa fraction was most prominent. Both non-labelled ivermectin and non-labelled retinol at 200-fold molar excess reduced binding of [3H]-ivermectin to all & fractions. These data indicate that the putative Onchocerca RBP has an approximate molecular weight of 19·7 kDa, that retinol also binds to 3 additional fractions non-specifically, that the pattern of binding of ivermectin to adult worm material is quantitatively and qualitatively different from the binding exhibited by retinol, and that ivermectin interferes with the binding of retinol to the 19·7 kDa Onchocerca protein.

The technique of allozyme electrophoresis was applied to species of Onchocerca from cattle to increase the number of enzyme loci established and therefore provide a genetic basis for a rational species-level taxonomy. Twenty-three enzyme loci were established and provided unequivocal genetic evidence for the taxonomic validity of Onchocerca gibsoni, O. gutturosa and O. lienalis. Furthermore, the diagnostic enzyme markers detected form the basis for identification of life-cycle stages, individuals and species and population structure analyses.

Testicular examination of male Onchocerca dukei and German O. tarsicola revealed five pairs of chromosomes, which is typical of most filariae. In contrast to German O. tarsicola, specimens from Sweden had only three pairs of chromosomes.

Trials were carried out to study the humoral immune response of blackflies to filariae following infection using the intrathoracic injection technique. An induced 66 kDa protein was abundant in the haemolymph of the European species Simulium ornatum following infection with bovine Onchocerca lienalis. This protein was apparently at higher concentrations in the haemolymph of sham-inoculated flies, i.e. flies that received sterile medium without the parasites. A molecule of the same size was also observed in the haemolymph of infected S. damnosum s.l. following infection with human O. volvulus or bovine O. ochengi. However, the level of this protein was lower in blackflies injected with microfilariae of bovine O. dukei. Unlike O. volvulus and O. ochengi this species is not transmitted by S. damnosum s.l. under natural conditions. No such reaction was observed if the African blackflies had received a sham inoculation. Feeding experiments with wild-caught nulliparous S. damnosum sl. on Onchocerca-infected cattle supported the results of the injection trials. The 66 kDa protein could only be found in the haemolymph of specimens infected via a blood meal. This 66 kDa molecule was identified as phenoloxidase. It appeared in the haemolymph due to the activation of the prophenoloxidase system following the filarial infection and we hypothesize that it may be sequestered by the parasites, as part of a non-self recognition system.

The behavioural responses of Onchocerca lienalis microfilariae (mf) to tissue factors of the surrogate black fly host, Simulium vittatum, were studied using a novel in vitro bioassay. Mf accumulated towards thoracic tissues to a density 4 times higher than towards abdominal tissues, despite the larger surface area and volume of abdominal tissues. Mf migrated toward thoracic tissues regardless of whether or not contact with thoracic tissues was possible. Therefore, mf directed themselves toward the thorax rather than arresting their movement after they make contact with thoracic tissues. Chemical cue(s) provided a principal guidance for mf to locate thoracic tissues. Mf lost their ability to differentiate thoracic and abdominal tissues following addition of thoracic attractant(s) to excised abdomens and reversed their differential response when excised thoraces were depleted of chemical cue(s). Mf did not respond to salivary gland product, but to other thoracic factor(s). Intact thorax that was connected to 1–2 abdominal segments attracted considerably less mf than excised thorax. However, intact thorax attracted more mf than excised abdomen. Abdominal tissue size did not affect thorax attractiveness. Involvement of a large molecular weight protein(s) was suggested by the high attractiveness of the 100 kDa Centricon concentrator retentate and complete precipitation of attractant(s) by ethanol.

Multiple parasitic infections have become increasingly recognized as a result of improvements in laboratory diagnosis and a growing population of immunocompromised individuals. This review examines the principles of chemotherapy in groupings of multiple infections which are of particular clinical significance.

The time-course of the humoral immune response of female blackflies after a challenge with bacteria, different Onchocerca microfilariae species, bacterial endotoxin and microfilarial extract was investigated. Strong bacteriolytic and growth inhibition activities against the Gram-positive bacterium Micrococcus luteus were induced by all agents. Specific differences were found in activity levels and time-course. Notably the endotoxin lipopolysaccharide (LPS) induced a very early, profound bacteriolytic and antibacterial response, which declined within a day after injection. In contrast, the bacteriolytic activities after Escherichia coli D31 and Onchocerca microfilariae infections were lower, but remained elevated over the observation period of 4 days. The bacteriolytic activity was correlated to a haemolymph protein with a molecular weight of around 14 kDa. Anti-Gram-positive activity in the E. coli infected group appeared within the first 6 h. However, it took 4 days in the microfilarial infected blackflies to reach significant levels. The active agent was identified to be a peptide with a molecular weight of around 4–4.5 kDa. Activity against the Gram-negative bacteria E. coli was detected in blackflies injected with E. coli D31, O. dukei microfilariae and microfilarial extract on days 1 and 4 after injection. The immune response in S. damnosum s.l. naturally infected via a bloodmeal on cattle supported the findings of the experimental infections. Similarities of the immune response kinetics between bacterial and filarial infections suggested that intracellular Wolbachia bacteria, released from microfilariae, could be responsible for the antibacterial response. This is supported by the observation that the induction of an immune response in the Drosophila melanogaster mbn-2 cell line by the filarial extract is blocked by polymyxin B, which forms inactive complexes with bacterial LPS.