Onchocerca ochengi:: morphological identification of the L3 in wild Simulium damnosum sl, verified by DNA probes

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 Ngaoundere 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 mu m), more slender (diameter = 19.3 mu m = 2.6% of body length) and had a relatively shorter tail (4.9% of body length) than those of O. volvulus (680 mu m, 20.5 mu m, 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 Ngaoundere, but were even longer (785 mu m). 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 non-hybridization 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.