The model of Onchocerca lienalis microfilariae (mf) injected into inbred CBA/Ca mice was studied for its usefulness as an additional primary/secondary drug screen for onchocerciasis. Invermectin, DEC, suramin, flubendazole, mebendazole, levamisole, Mel W, furapyrimidone, metrifonate, amoscanate and the new Ciba-Geigy compounds CGP 6140, CGP 20'376 and CGI 17658 all significantly reduced levels of mf at a dose of 5x100 mg/kg or less. An early dosing protocol, on days 3–7 after infection, was found to be generally more effective than dosing on days 11–15, followed by necropsy on day 18. In some cases there were important differences in levels of drug activity depending on whether the drug was administered by the subcutaneous or oral route, indicating that new compounds should be tested via both routes. Ivermectin was by far the most active compound examined, virtually clearing mf from the skin at a dose of 5x0.0063 mg/kg and producing a significant mf reduction (63.5%) at 5x0.008 mg/kg following subcutaneous administration. In comparison, DEC was much less active, producing a 32.4% mf reduction at 5x25 mg/kg ranging up to a maximum of 72% reduction at 5x100 mg/kg. CGI 17658 was the most active compound examined next to ivermectin, almost 100% effective against skin mf at a dose of 5x6.25 mg/kg via the oral route while being less effective via subcutaneous administration (65% reduction). The lowest effective dose examined was 5x3.13 mg/kg (per os) which reduced mf levels by 64%. CGP 20'376 was also very active, resulting in a 46% (subcutaneous) and 62% (per os) reduction at a dose of 5x6.25 mg/kg. This mouse model has clearly identified all the known microfilaricides examined and also, to a lesser extent, those compounds considered to be principally macrofilaricides. We believe it has value as an additional drug screen for onchocerciasis, which will enable the evaluation of novel compounds against skin-dwelling Onchocerca mf at the primary/secondary level, providing complementary information to new in vitro screens using adult Onchocerca.

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.

The viability and drug responses of cryopreserved adult Onchocerca have been examined in vitro. Male worms were cryopreserved in liquid nitrogen (-196 degrees C) using ethanediol as a cryoprotectant in a 2-step incubation procedure. After thawing, 85-90% of O. gutturosa males were normally motile. These motile worms were evaluated for viability using 4 measurements (long-term motility/survival in culture; [U-14C]adenine uptake and leakage; glucose utilization; MTT-formazan colorimetry) and were no different from unfrozen controls. Subsequent experiments demonstrated that the motility responses of cryopreserved worms exposed to the antifilarial drugs ivermectin, CGP 6140 and levamisole were virtually identical to unfrozen controls. Some success was also obtained with this technique in cryopreserving O. volvulus males, with 2 thawed specimens surviving in culture for 93 and 106 d respectively. Following collagenase isolation, female worms were cryopreserved in medium +10% serum without protectant at -79 degrees C. A batch of 8 female O. gutturosa were all motile when thawed 14 d later, with a mean survival time (based on 5 specimens) of 71 d (range 60-90). However, a batch of worms transferred from -79 degrees C to -196 degrees C were badly damaged when thawed. Female O. volvulus were cryopreserved at -79 degrees C in Guatemala and sent by air freight on solid CO2 to the UK. Most specimens were active when thawed. Survival of motile specimens ranged from 7 to 272 d in culture. It is concluded that these techniques are of practical value for the storage and transportation of adult Onchocerca.

The isoenzyme profiles of five isolates of the supposed 'species' of Trichinella, T. nativa, T. spiralis and T. nelsoni were compared. Four enzymes (AK, PGM, MPI and GPI) gave good resolution and clearly differentiated T. Spiralis from the other two species. T. nativa and T. nelsoni had similar isoenzyme patterns; the two separate isolates of T. nativa and T. spiralis used gave similar results, thus indicating the validity and the reproducibility of the technique. The value of enzyme electrophoresis for specific and subspecific classification of Trichinella is discussed and compared with the more traditional methods of taxonomy which have failed to resolve the controversy surrounding speciation.