The differentiation of microfilariae of Onchocerca lienalis in vitro.
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This study evaluated the usefulness of parasite-specific IgG and IgG isotype responses for diagnosing early or pre-patent onchocerciasis in children using sandwich ELISA. The children (n = 199) were aged between 5 and 12 years and living in a meso-endemic area of northern Nigeria. Only five had detectable skin microfilariae. The mean optical density (OD) values of children from the study area were significantly higher than those of normal controls' sera (n = 10, p < 0.01), except for IgG2. There were 145 (73%) children positive for total IgG, 161 (81%) for IgG1, 68 (34%) for IgG3 and 187 (94%) for IgG4 antibodies. The mean OD values of all antibodies tended to increase with age and peaked in the 9-10-year age group, except in the case of IgG3 which peaked at 11-12 years. IgG4 proved most sensitive compared with IgG, IgG1 and IgG3 in all age groups, and was significantly different (p < 0.05). Analysis by sex showed no significant difference between the boys and girls. We conclude that IgG4 serology is a useful diagnostic tool in the assay of early or pre-patent onchocerciasis in children.
We have cloned and characterized the gene for an immunodominant antigen of O. volvulus that is recognized by the sera of 96% of patients with onchocerciasis. Its 1.2-kb mRNA constitutes 0.3% of adult worm poly(A)+ RNA and its cDNA sequence reveals that it is not a highly conserved structural protein such as actin or tubulin. Similar but not identical genes occur in the genomes of related filarie, Brugia malayi and Dirofilaria immitis. The recombinant antigen has both immunodiagnostic and immunoprophylactic significance.
In persons with onchocerciasis, topical application of the anthelminthic diethylcarbamazine (DEC) induces clinical and histologic responses similar to acute papular onchodermatitis, including recruitment of eosinophils to the skin. To determine whether the eosinophil chemokine eotaxin is likely to be associated with eosinophil recruitment in onchodermatitis, DEC was applied to a 5-cm2 area on the skin of infected persons, and biopsies were taken from lesions 24 h later. Histologic analysis showed elevated dermal and epidermal eosinophils compared with tissue from an adjacent (untreated) site. Reverse transcription-polymerase chain reaction showed that eotaxin gene expression in DEC-treated skin was elevated 2- to 17-fold compared with control tissue. Eotaxin immunoreactivity was noted in mononuclear cells and eosinophils in the perivascular region of the dermis and in lymphatic and vascular endothelial cells. Together, these observations are consistent with a role for eotaxin in recruitment of eosinophils to the dermis in early stage onchocercal skin disease.
The standard assay for onchocerciasis diagnosis is microscopic detection of parasites in skin snips. Skin snipping is painful and may potentially transmit bloodborne infections. Thus, an alternative method for the diagnosis of onchocerciasis that does not require skin snipping is needed. A polymerase chain reaction (PCR)-based assay was shown to detect the presence of parasite DNA in superficial skin scrapings. Detection of parasite DNA in both skin snips and skin scratches was found to be more sensitive for detecting low-density infections than was microscopic examination of skin snips. The skin scratch PCR assay is minimally invasive and painless and does not present the risk of transmitting bloodborne infections. These properties make the skin scratch an attractive alternative to the skin snip for detecting O. volvulus infection.
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Laboratory developmental studies were carried out on Simulium bidentatum (Shiraki) under constant air and water temperatures (7, 10, 13, 16, 19, 22, 25, and 28 degrees C). The median time required for female flies to complete oocyte maturation after blood feeding decreased from 8 to 1.5 d and the median time from blood feeding to oviposition decreased from 16 to 3 d as air temperature increased from 7 to 28 degrees C. Air temperatures from 16 to 22 degrees C were most suitable for oviposition. An average of 61.4% of eggs hatched. Egg developmental period decreased from 17 to 4.5 d when water temperature increased from 7 to 19 degrees C and decreased to 3.5 d when water temperature was between 22 and 28 degrees C. Larval and pupal development occurred at water temperatures > or = 13 degrees C, but was most successful from 16 to 22 degrees C. Seven (in 16 lots) or 6 instars (in 1 lot) were indicated by using the Dyar rule. We conclude that S. bidentatum is adapted to moderate weather conditions in spring and autumn and that approximately 7 generations of this species occur per year in Oita, Japan.
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Migration of human peripheral blood granulocytes in response to microfilariae of O. volvulus was demonstrated using modified Boyden chambers. Granulocyte migration was significantly enhanced when microfilariae were preincubated with heat-inactivated immune serum (delta IS), then added to a fresh serum source (P less than 0.025). This effect was not seen when microfilariae were incubated in medium alone, in delta IS alone, in delta IS plus C4-deficient guinea-pig serum, or in fresh serum alone. There was no significant difference between the response of cells from O. volvulus-infected donors and that of cells from normal volunteers. Likewise, there was no significant difference between the migratory response seen toward nodule- versus skin-derived microfilariae. These results suggest that the host inflammatory response to O. volvulus microfilariae is mediated in part by chemotactic factors generated by antibody and complement interaction with the organism and, furthermore, that these factors are product(s) of classical complement pathway activation.
The prevalence of IgG antibodies to three recombinant O. volvulus antigens, OvMBP/10, OvMBP/11 and OvMBP/29 was determined in a group of 94 microfilaria positive (mf+) individuals resident in the hyperendemic onchocercal area of Esmeraldas Province, Ecuador. Clone OvMBP/11 was the antigen most frequently recognized by patients sera followed by OvMBP/10 and OvMBP/29. When a cocktail of the three recombinant antigens was used the proportion of positive sera increased to 100%. Antibody responses to the fusion partner maltose binding protein (MBP) were low in comparison with those to the cloned antigens and no correlation of responses between individual antigens was observed. The relative level of antibody response to each of the clones in the cocktail varied between individuals. The distribution of IgG responses to OvMBP/11 was bimodal and those to OvMBP/29 and OvMBP/10 were positively and negatively skewed, respectively. When the three recombinant antigens were used in combination this variation was minimized and the pattern of responses showed a normal distribution as was also seen to crude O. volvulus antigen. The cocktail of recombinants thus offers excellent diagnostic sensitivity in combination with the parasite specificity demonstrated previously.