The transmission of bovine Onchocerca spp. in Britain was investigated by studying the Ceratopogonidae and Simuliidae which attacked bait cattle between April and October at two sites in North Wales, where either Onchocerca lienalis and O. gutturosa (Cynwyd), or O. lienalis alone (Pentrefoelas) occurred. Based on the relative abundance of each species, their seasonal variation in abundance and preferential feeding sites on cattle, the presence of Onchocerca sp. infective larvae and the development of patent infections in bait cattle, vectors were identified as Simulium ornatum s.1. at Pentrefoelas and S. reptans at Cynwyd (0·5% and 1·5% of parous flies infective, respectively). There was no significant difference between the lengths of the infective larvae from vectors at either site (Pentrefoelas: mean = 543·5 μm, SD = 29·45; Cynwyd: mean = 550·86 μm, SD = 35·26; p > 0·1), which were consistent with descriptions of O. lienalis. The identity of a vector for O. gutturosa was not determined and the role of certain candidates is discussed. In this respect, the absence of Culicoides nubeculosus was notable. Transmission of Onchocerca sp. was limited to a short period of about 8 weeks, by a combination of vector abundance and parasite uptake from dermal skin layers, and a mean daily temperature requirement of 15°C or more for complete parasite development in the vector. The effect of this temperature restriction in relation to the geographical distribution of the two species is discussed.

An in vitro system for chemotherapeutic research using adult male Onchocerca gutturosa has been developed as a model for O. volvulus. Using a culture system consisting of medium MEM+10% heat inactivated foetal calf serum (IFCS)+LLCMK2 (monkey kidney) feeder cells in an atmosphere of 5% CO2 in air, we examined the effects of a range of antiparasitic drugs on worm motility. Ivermectin, levamisole, furapyrimidone, Mel W, chloroquine, metrifonate, flubendazole, amoscanate and the Ciba-Geigy compounds CGP 6140, CGP 20′376 and CGI 17658 either immobilized or significantly reduced motility levels at a concentration of 5x10−5M or less within a 7-day period. Worms were affected at very low concentrations by ivermectin (effective conc. to reduce motility levels to 50% of controls, 3.14x10−8M), levamisole (7.95x10−8M), CGP 6140 (8.87x10−9M) and CGP 20′376 (2.78x10−8M). Difficulties were experienced in accurately repeating the immotile endpoint for levamisole due to an inconsistent partial recovery of motility. Over a 7-day period diethylcarbamazine had little effect on motility levels, while suramin caused a slight increase in activity compared to controls at some timepoints. Subsequent experiments demonstrated some differences in drug efficacy depending on the presence or absence of serum and feeder cells in the culture system probably because of drug avidly binding to serum proteins. However, serum and cells were found to be essential ingredients of the culture system to maintain worms in good condition, indicating that new drugs should be evaluated both in the presence and absence of serum and cells. Comparisons were made between the responses of O. gutturosa and Brugia pahangi to certain drugs and these species were found to significantly differ in their sensitivities to ivermectin and a novel compound (Wellcome), indicating that Onchocerca parasites should be used wherever possible for compound identification and development intended for the treatment of onchocerciasis. The in vitro system described here, using male O. gutturosa, provides a basis for further research and a practical alternative to O. volvulus.

A series of experiments examined the effects of various media, serum supplements, gas phases and the incorporation of mammalian cell feeder layers on the survival on Onchocerca gutturosa adult worms in vitro. The survival of male worms was poor in all media tested that were not supplemented with inactivated foetal calf serum (IFCS), with improved but variable survival in media supplemented with 10–30% IFCS. Using a cell-free system in an atmosphere of 5% CO2in air, good results were obtained in medium NCTC 135 + 10% IFCS (median survival time 39 days, range 25–41). Marginally better survival was obtained with the same medium in an atmosphere of 95% N2/5% CO2 (medium 45 days, range 25–56)and with a 1:1 mixture of media NCTC 135 and IMDM+10% IFCS(medium 38 days, range 38–51). Survival was enhanced in culture systems which incorporated bovine kindney (MDBK) cells, bovine trachea (EBTR) cells and monkey kidney (LLCMK2) cells. Exceptionally long survival was obtained using medium MEM+10% IFCS+LLCMK2 cells under a gas phase of 5% CO2in air, in which male worms survived from approximately 6 to over 7 months. Under similar conditions, female worms were also maintained for periods of up to 6 months and 5 out of 18 specimens released microfilariae into the culture system. The long-term culture described in this study will be useful for basic biochemical, chemotherapeutic and immunological studies in vitro.