Onchocerca gutturosa and O. volvulus: studies on the viability and drug responses of cryopreserved adult worms in vitro

Abstract

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.

Automated Imaging and other developments in whole‐organism anthelmintic screening

“…Simple indicators of metabolic activity such as the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide), Alamar blue (resazurin) and acid phosphatase activity have been widely used to assess viability of infectious organisms including several helminth species. MTT, which allows a simple colorimetric readout of redox enzyme activity was first applied to filarial nematodes (72) and has since been adapted to a variety of helminths (28,39,51,(73)(74)(75)(76). Alamar blue, the basis of a similar colorimetric/fluorometric assay has been adapted to drug screening for Schistosoma species (32,77) and T. muris (51) and the acid phosphatase test for screening using T. muris (51), Taenia crassiceps (78) and H. bakeri (79).…”

“…Nevertheless, whole‐organism screens have several advantages over target‐based screening in that they establish that compounds can reach their chemical space and desired target and are not constrained to a single target within these complex eukaryotes, for which the majority of possible targets including those for known anthelmintics have yet to be identified . For human helminths, the adult worms are the prime targets for chemotherapy, and in vitro screens using adults have been implemented . Although such screens offer advantages over in vivo testing, they are inevitably low throughput due to the size of the adult worms and reliance on experimental animals for their provision.…”

Challenges in drug discovery for novel antifilarials

“…As research progressed towards better understanding of parasite morphology, the elucidation of the host -parasite relationships and early drug studies, further refinements in culture conditions were introduced in subsequent years [66][67][68][69][70][71][72][73][74][75] . Testing compounds for macrofilaricidal activity is a recent application of adult parasite in vitro culture [76,77] .…”

The anterior central nervous system of male Onchocerca volvulus (Nematoda: Filarioidea) as a target for chemotherapy: results of an electron microscopy study

“…This heterogeneity complicates the distinction of changes in worm tissues and worm metabolism that are due to age processes from those caused by treatment (Striebel 1988) or other intervention steps ). Since the histological evaluation of drug ecacy is by far the most important and useful parameter in phase II clinical trials involving the target organism Onchocerca volvulus (BuÈ ttner 1985; Awadzi et al 1991Awadzi et al , 1995Duke 1991) and is of comparable importance in preclinical experiments with rodent, cattle, and in vitro models (Franz et al 1987(Franz et al , 1990Townson et al 1987Townson et al , 1989Franz 1988;Townson 1988;Strote et al 1990aStrote et al , 1993, a distinction of these eects is decisive. Working at the light microscope level with the predictive cattle model O. gibsoni, Striebel and Copeman (1990) emphasized that tissues in the most anterior part of these ®larial worms are those best preserved as compared with the tissues and organs in the posterior part.…”

“…We therefore decided to study the ®ne structure of the anterior nerve ring after incubation of viable adult worms with known and new compounds in vitro. This inevitable experimental situation with the host-speci®c target parasites has been proven to be of predictive value in terms of the activity of standard compounds and new promising therapeuticals (Nowak et al 1987;Townson et al 1987Townson et al , 1989Strote et al 1990aStrote et al , b, 1993.…”