There is no published account which allows the morphological discrimination of microfilariae of Onchocerca volvulus and M. ozzardi from each other. However, they occur together in parts of Brazil and Venezuela, and presumably there is always the possibility that migration could establish new sympatric populations in the future. The objective of this study was to evaluate simple morphological characters that might be used for species-diagnosis of microfilariae. The conclusions were that the location of microfilariae in the blood or skin, the body size and the nucleation of the nerve ring are expected to be useful first indications of species identity, but cannot be used for confident diagnosis. The structure of the cephalic armature (stained with alcian blue) seems to be species specific, but is of limited application because it is often difficult to see. However, the pattern of nucleation of the tail (as expressed by the ratio of the length of the terminal nucleus compared with the length of the tail space) is distinctive and is expected to be diagnostic.

Onchocerca volvulus is a pathogenic human filarial parasite which, like other helminth parasites, is capable of evading the host's immune responses by a variety of defense mechanisms which are likely to include the detoxification and repair mechanisms of the enzyme glutathione S-transferase (GST). In this study, we show that one of the previously described GSTs from O. volvulus appears to possess the characteristics of a secreted enzyme. When the complete O. volvulus GST1 (OvGST1) sequence presented here is compared with those of other GSTs, 50 additional residues at the N terminus are observed, the first 25 showing characteristics of a signal peptide. This is consistent with the N-terminal sequence data on the native mature enzyme which begins at amino acid 26, based on the deduced protein sequence from the cDNA. The native protein, without the signal peptide sequence, possesses a 24-amino-acid extension not present in other GSTs. The deduced amino acid sequence of the OvGST1 cDNA clone was shown to possess four potential N-glycosylation sites. Digestion of O. volvulus homogenate with endoglycosidase, followed by detection of OvGST1 with specific antibody, indicated that the enzyme possesses at least two N-linked oligosaccharide chains. Gel filtration of the Escherichia coli-produced recombinant OvGST1 showed that it is enzymatically active as a nonglycosylated dimer. OvGST1 is found in the media surrounding adult worms maintained in culture, indicating that, in vitro, this enzyme is released from the worm. The strongest immunostaining for OvGST1 was observed in the outer cellular covering of the adult worm body, the syncytial hypodermis, especially in the interchordal hypodermis, where the peripheral membrane forms a series of lamellae which run into the outer zone of the hypodermal cytoplasm.

The effects of single and multiple doses of ivermectin on mortality and morphology were assessed in over 700 female Onchocerca volvulus worms and the effects on embryogenesis were assessed in 490. Nodules were surgically removed from Sierra Leoneans recruited from a double-blind placebo controlled study of ivermectin given at six-monthly intervals. Nodules were digested in collagenase to isolate whole adult worms. After four or five doses of ivermectin there were significant increases in the numbers of discoloured and calcified worms and possibly a trend towards increased mortality, but this was not seen consistently. There was no evidence of a prophylactic effect of the drug. Worms were then homogenised and embryograms constructed. A single dose of ivermectin produced large numbers of degenerating intrauterine microfilariae, but embryonic development occurred normally. After multiple doses we observed almost complete cessation of embryogenesis, with a highly significant decrease in the numbers of viable multicellular embryonic stages, while oocytes appeared to be produced normally. Development is probably impeded at the single cell stage, possibly because of reduced fertilization. In planning the future role of ivermectin as a control measure for onchocerciasis it is crucial to determine if these effects on embryogenesis are reversible.

Immunization of chimpanzees with radiation-attenuated infective 3rd-stage larvae (L3) of Onchocerca volvulus did not induce strong protective immunity against a subsequent challenge infection; only 1 out of 4 immunized animals remained non-patent (i.e. microfilariae-negative) after challenge, and may have been protected. However, during immunization and before challenge, a broad range of adult O. volvulus-derived antigens (OvAg) and also uterus-derived OvAg were recognized by circulating antibodies; moreover, the repertory of antigens recognized increased further in subsequently patent animals after challenge, particularly in the range of M(r) 12-42 kDa. In the immunized and non-patent chimpanzee, by contrast, serological recognition of uterus-derived OvAg with M(r) 14 kDa and 105 kDa disappeared by 19 months post-challenge (p.c.). During immunization, Acanthocheilonema viteae L3 antigens of M(r) 11-12 kDa were strongly recognized only by the non-patent animal, suggesting that recognition of these antigens may have supported resistance to the subsequent challenge infection. In immunized chimpanzees, a substantial increase in the cellular reactivity to OvAg was induced; this, however, declined by 19 months p.c. to levels similar to those seen prior to immunization. At that time, 3 out of 4 immunized animals were patently infected. The effect of exogenous cytokines on in vitro-reactivity of PBMC to OvAg was examined. Addition of exogenous IL-2 alone, IFN-gamma alone, and IFN-gamma in combination with IL-2, did not augment net cellular responses to OvAg by PBMC from infected and control chimpanzees. In the presence of IL-4 alone, IL-6 alone, IL-2 with IL-4, IL-2 with IL-4 and IFN-gamma, or IL-2 with IL-4 and IL-6, the net cellular reactivity to OvAg increased significantly in patent chimpanzees and reached levels similar to non-patent animals. Thus, non-patent chimpanzees maintain high cellular reactivity to OvAg and in vitro cellular unresponsiveness to OvAg on the part of patent chimpanzees is reversible after addition of several cytokines which act individually or synergistically.

A pleomorphic neoplasm (PN) is described from sections of Onchocerca volvulus worms in nodules excised from Cameroonian patients. PN is confined to older, non-fecund, female worms, and those classed as moribund/dead. It is mainly composed of small, roundish, basophilic cells of diverse sizes, often forming a 'rosette' pattern around amorphous eosinophilic centres. The cells have a high nuclear/cytoplasmic ratio and up to 2-3 mitoses/high-power field; some become grossly enlarged, highly polymorphic and contain large, irregular blocks of chromatin. The eukaryotic PN cells first appear posteriorly in the pseudocoelom, probably from ovarian cells; they spread anteriorly, invading or compressing the uteri. Ivermectin treatment increased the prevalence PN from 3.7% of 1422 female worms in 637 patients before treatment to 17.5% of 1134 worms in 511 patients after 3 years treatment. Ivermectin at 400-800 microg/kg annually, or at 150 microg/kg or 400-800 microg/kg 3-monthly, over 3 years, did not increase the PN prevalence significantly, as compared with standard doses of 150 microg/kg annually. In other small series of African patients, PN prevalence increased in worms 2, 4, 6 and 10 months after ivermectin treatment; but there was no increase after treatment with amocarzine, albendazole or diethylcarbamazine and suramin. PN may partly account for the increased macrofilaricidal action of ivermectin on female O. volvulus in patients treated for 3 years at 3-monthly intervals.

There is no published account which allows the morphological discrimination of microfilariae of Onchocerca volvulus and M. ozzardi from each other. However, they occur together in parts of Brazil and Venezuela, and presumably there is always the possibility that migration could establish new sympatric populations in the future. The objective of this study was to evaluate simple morphological characters that might be used for species-diagnosis of microfilariae. The conclusions were that the location of microfilariae in the blood or skin, the body size and the nucleation of the nerve ring are expected to be useful first indications of species identity, but cannot be used for confident diagnosis. The structure of the cephalic armature (stained with alcian blue) seems to be species specific, but is of limited application because it is often difficult to see. However, the pattern of nucleation of the tail (as expressed by the ratio of the length of the terminal nucleus compared with the length of the tail space) is distinctive and is expected to be diagnostic.

In order to assess the prevalence of the cattle filaria Onchocerca ochengi in onchocerciasis vectors (Simulium damnosum s.l.) in North Cameroon, we searched for a means to morphologically identify its developing larvae, which closely resemble those of O. volvulus. To this end microfilariae of the 2 Onchocerca species were isolated from slaughter cattle in Ngaoundéré and injected into neonate Simulium species. Whereas the early developmental stages (sausage stage, L2 and pre-infective larva) were indistinguishable, the infective larvae (L3) of O. ochengi were longer (median: 740 microns), more slender (diameter = 19.3 microns = 2.6% of body length) and had a relatively shorter tail (4.9% of body length) than those of O. volvulus (680 microns, 20.5 microns, 3.0% and 5.8% respectively). The tail of O. ochengi L3 was thick and rounded, whereas it was slightly tapering in O. volvulus L3. O. ochengi L3 produced by feeding flies on infected cattle in a different area in North Cameroon (Sora Mboum) showed the same features as intrathoracically produced O. ochengi L3 from Ngaoundéré, but were even longer (785 microns). On the basis of the differences in length, relative diameter, length of the tail and shape of the tail, a simple key for the separation of O. volvulus and O. ochengi L3 was elaborated, and 248 L3 found in wild S. damnosum s.l. were separated into 'O. ochengi'(160 L3) and 'O. volvulus'(88 L3) following this key. Sequential dot blot hybridization of each of the 248 larvae with a DNA probe which reacts with O. ochengi and O. volvulus but not with other Onchocerca species (pOo5/1) and with an O. volvulus-specific DNA probe (pOv12) revealed that the morphological identification had been correct in 86-91% of the cases. Only a small proportion (6-9%) of the dot blots did not react with either probe. Since this proportion was equal in experiments using experimentally produced L3 and in experiments using wild L3, the nonhybridization was certainly due to a loss of L3 during washing of the filters and not due to the presence of other unknown L3 species resembling O. volvulus and O. ochengi. Our study shows that in Cameroon it is possible to identify O. volvulus and O. ochengi infective larvae during routine fly dissections by morphology alone.

The population structure of Onchocerca volvulus macrofilariae was studied in villages of central Cameroon where onchocerciasis is hyper-endemic. One nodule selected at random was removed from each of 576 adult males, and examined by histology. The numbers of male and female worms/nodule, and the status of the female worms (fecund, non-fecund, or dead) were recorded. The observations were analysed to evaluate whether the mean numbers of worms of each category varied in relation to the patient's age, the level of endemicity in his village, the anatomical localization of the nodule, the weight of the nodule, and the total number of palpable nodules harboured by the patient. The results obtained were very similar to those reported from West Africa. The mean numbers of dead female worms/nodule were relatively high in the villages with the lowest levels of endemicity. The mean numbers of fecund females and of live males were significantly higher in the nodules located around the knees. These results provide information which might be useful in modelling the population dynamics of O. volvulus, and also in the context of trials of any potentially macrofilaricidal drugs.