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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.

In North Cameroon, the vector of Onchocerca volvulus (causative agent of human onchocerciasis) also transmits 2 filariae of animals: O. ochengi from cattle and O. ramachandrini from wart hogs. In order to assess the qualitative and quantitative roles of these 'animal filariae' in the epidemiology of O. volvulus, the transmission of the 3 parasites was measured in 2 villages and related to the endemicity of human onchocerciasis. In Galim, a cattle-farming Guinea savanna village where wild animals are rare, the overwhelming majority of all filarial infections found in the Simulium damnosum s.l. vectors throughout the year were O. ochengi (89%). The remaining infections were mainly O. volvulus (10.5%), and a few O. ramachandrini (0.5%). In Karna, a crop-farming Sudan savanna village where cattle are rare, but wild animals common, flies were also more frequently infected with animal filariae than with the human parasite. In the dry season, when nomadic cattle are present, 54% of all infections were O. ochengi, 36% O. volvulus and 10% O. ramachindrini. In the rainy season, when the cattle move away, flies were mainly infected with O. ramachandrini (52% of all infections) and secondly with O. volvulus (48%). In Karna, the relationship between the Annual Transmission Potential (ATP) of O. volvulus and its prevalence in the human population conformed to other onchocerciasis foci, in that a moderate ATP led to hyperendemic onchocerciasis. In Galim, however, a 7-fold higher O. volvulus-ATP (caused by a very high biting rate of the flies) contrasted with a strikingly low endemicity of onchocerciasis. Since, at the same time, in Galim the transmission of O. ochengi (measured on man) was very high (15,000 L3/fly collector/year), we hypothesize that the reduced endemicity of onchocerciasis in Galim is due to 'natural heterologous vaccination' by the large annual number of O. ochengi-L3, inoculated into man by anthropo-boophilic S. damnosum s.l. The importance of micro-epidemiology for the understanding of the interlinkage of human and animal onchocerciasis is discussed.

In the savanna areas of tropical Africa, cattle are frequently infected with the filaria Onchocerca ochengi. This parasite is closely related to Onchocerca volvulus, the causative agent of human onchocerciasis (river blindness), and is capable of developing in the same vector, Simulium damnosum s.l. In North Cameroon, where both O. ochengi and O. volvulus are endemic, we carried out a field study (reported in this and 2 following papers) to examine to which extent the transmission of the 2 parasite species overlap and what influence this has on the epidemiology of human onchocerciasis. In this paper we report our experiments to determine which of the S. damnosum species in North Cameroon act as vectors of O. ochengi, how efficiently they do so and whether other Simulium species play a vector role. To this end, infected cattle were exposed near 5 rivers in different geographical areas. Among 14 Simulium species identified as aquatic and/or adult stages at these rivers, only 6 (S. squamosum, S. damnosum s.s., S. sirbanum, S. bovis, S. wellmanni and S. hargreavesi) were found to bite cattle in important numbers in at least 1 of the sites. The 3 species of the S. damnosum complex were all capable of ingesting microfilariae (mf) of O. ochengi and developing a proportion of them to infective larvae (L3). Whereas S. squamosum and S. damnosum s.s., the prevailing vectors in the Guinea and Sudan savanna respectively, showed a high vector competence (17% of ingested mf developed to L3), S. sirbanum, which was much rarer in both areas, appeared to have a much lower susceptibility (2%). Other boophilic Simulium species were only seen in certain sites and seasons, being either incapable of ingesting important numbers of O. ochengi mf from body regions where these mf were abundant (S. bovis, S. hargreavesi); not able to support the development of ingested mf to L3 (S. wellmanni), or bit cattle preferentially in the ears, where O. ochengi mf do not occur (S. hargreavesi). We conclude that in North Cameroon members of the S. damnosum complex are the only important vectors of O. ochengi, with S. squamosum and S. damnosum s.s. being the main vectors.

Filarial larvae resembling Type D (Duke, 1967), which are common in the Simulium damnosum s.i. vectors of human onchocerciasis ("riverblindness") in several parts of West Africa, were dissected from wild-caught flies in north Cameroon and examined morphologically. This was done in order to establish a possible synonymy with infective larvae (L3) of 2 recently discovered Onchocerca species of wart hogs (Onchocerca ramachandrini Bain, Wahl, and Renz, 1993 and Onchocerca sp. Wahl and Bain, 1995), which had been found to resemble Type D. After dissection of approximately 1,700 S. damnosum s.1., 13 Type D-like larvae were recovered from 12 infected flies. Their morphology corresponded to O. ramachandrini.

A simple method to obtain large numbers of viable infective larvae (L3) of the human filarial parasite Loa loa in an endemic country is described: wild vector flies (Chrysops silacea and C. dimidiata) were caught in large numbers, sealed into pockets of mosquito netting, gently crushed on a glass plate and incubated in a funnel with medium ("Baermann technique"). 91-94% of the L3 actively left the crushed flies and accumulated in the funnel tip after 15 (80%) to 45 minutes (95%). During 111 catching days over a 12 month period, a single fly collector provided approximately 13,000 flies and 23,500 L3. The method is also considered useful for assessing loiasis-transmission, for example before and after mass treatment campaigns.

Four wart hogs (Phacochoerus aethiopicus) examined in the Sudan savanna of North-Cameroon were all found infected with two types of skin microfilariae. One was O. ramachandrini Bain, Wahl and Renz, 1993, the adult worms of which live in the subcutaneous tissues of the feet. The other, smaller type belongs to a new Onchocerca species, the adult worms of which were not yet found. O. ramachandrini-microfilariae were evenly distributed across the whole body surface, those of Onchocerca sp. were concentrated on the back. The two species of microfilariae were isolated from an infected hide separated under the dissecting microscope and injected into the thorax of pupae-hatched S. squamosum and S. damnosum s.slr. females. Both filariae developed in both flies at high rates (33-47% of injected microfilariae) and without pathological forms to infective larvae L3). Both L3-species had a caudal tip, were long, slender and very motile and had a conspicuous glandular oesophagus. L3 from O. ramachandrini-microfilariae had a long glandular oesophagus (55% of total L3 length), a round head and measured an average of 955 microns long and 19.2 microns wide. L3 from the other microfilaria-species were shorter (845 microns, P < 0.001) and thinner (16.7 microns, P < 0.001) and had a shorter glandular oesophagus (36%, P < 0.001), a shorter tail (P < 0.01) and a conical head. Both L3-species, by their caudal tip, their long and slender silhouette, their great motility and their conspicuous glandular oesophagus resemble non-O. volvulus filarial L3 known, since many years, to occur in "wild" S. damnosum s.l. in Cameroon (Type D larvae, Duke, 1967) and in Liberia (Agamofilaria Type VI, Voelker and Garms, 1972). During our study, L3 such larvae were found in 12 wild S. damnosum s.l. from two geographically different areas of North Cameroon and all identified as O. ramachandrini. The excellent development of the two Onchocerca species from the wart hog in S. damnosum s.l. after artificial infection, and the identification of all recently examined wild Type D larvae as O. ramachandrini suggest that S. damnosum s.l. is a natural vector of O. ramachandrini and that most (if not all) of the Type D larvae in onchocerciasis vectors in North-Cameroon originate from wart hogs.

"Wild" Simulium bovis females were collected after they had taken blood on a bait oxen with skin microfilariae (mf) of Onchocerca dukei and Onchocerca ochengi. Flies ingested O. dukei mf in high numbers (13.1 mf per blood fed fly) but rarely mf of O. ochengi (3 mf in 60 flies). In 32 flies dissected 14 to 17 hours after the blood meal, a total of 130 O. dukei mf but no of O. ochengi had migrated to the thorax. Six to 9 days after the blood meal, a total of 203 third stage larvae (L3) were found in 61 surviving flies. Invasive larvae from the head measured 738.0 x 17.8 microns and resembled those of O. dukei obtained by intrathoracic injection of mf in a previous study, but were shorter and thinner than O. ochengi L3. Thus, S. bovis can be seen as an efficient natural vector of O. dukei. Its role in the transmission of bovine and human onchocerciasis in North Cameroon is discussed.

Microfilariae (mff) of the savanna and forest strains of Onchocerca volvulus (Leuckart) were injected intrathoracically into adult females of Simulium damnosum Theobald sensu stricto, S.sirbanum Vajime & Dunbar, S.squamosum Enderlein and S.mengense Vajime & Dunbar. Nine days post infection (pi) 27-29% of the savanna mff and 31-38% of the forest strain had developed to third-stage larvae (L3), irrespective of the fly species, size or injection dose (5, 10 or 15 mff). Savanna flies supported the development of forest O.volvulus better than forest flies, in contrast to the results after per os infections. Therefore, in these four species of the S.damnosum complex from Cameroon, the peritrophic membrane is considered to be the main factor limiting the success rate of microfilarial development following the ingestion of blood infections, while the fly's haemolymph and intracellular environment play minor roles.

Onchocerca microfilariae were isolated form the umbilicus and neck of 438 cow hides at the abattoir in Tübingen, F.R.G. The overall Onchocerca infection rate was 40.4%. The presence of Onchocerca lienalis and O. gutturosa microfilariae, which are difficult to distinguish by morphological criteria, was retrospectively demonstrated after artifically infesting Simulium ornatum and Culicoides nubeculosus and identifying the infective larvae recovered. Nine of 16 samples of umbilical microfilariae fed to C. nubeculosus through a latex membrane developed to O. gutturosa third stage larvae (L3). Six of seven umbilical samples injected into the thorax of S. ornatum yielded O. lienalis L3. In six infestation trials in which microfilariae were introduced both into S. ornatum and C. nubeculosus, O. lienalis L3 were recovered exclusively from simuliids, while O. gutturosa L3 developed only in midges. Of six umbilical skins tested by cross-infestation, one contained exclusively O. gutturosa microfilariae, four only O. lienalis microfilariae and one was infected with both species. Developmental success of O. lienalis microfilariae to L3 in S. ornatum following intrathoracic injection was 22% of the mean inoculum. O. gutturosa microfilariae, ingested by C. nubeculosus through a latex membrane, developed to L3 at a rate of 2.3% of the mean microfilarial uptake.