Viability of Onchocerca volvulus in vitro

Abstract

The use of a selective schedule of tests to identify a viable population of isolated adult Onchocerca volvulus (Nematoda: Filarioidea) has been investigated in a large worm population. The study was initiated to develop methodology appropriate to test new candidate macrofilaricides for their in vitro activity against O. volvulus. After removal from the host the viability of isolated intact parasites was estimated by assessing the motility indices of male worms, and the colorimetric quantification of the reduction of the bioreducible tetrazolium reagent XTT and lactate output by female worms. Additionally the motility of whole females and the movement of inner organs of female worms were scored quantitatively. These response parameters were used to sort the adult worms into viability groups at the start of the in vitro culture. The adult worms were then observed for 6 days and viability was assessed regularly during the culture period. At the end of the culture period, the reduction of the water-insoluble tetrazolium reagent MTT was used to determine the formazan formed by the entire male and female worms. The response parameters used at the start of the culture proved to be highly predictive for detecting viable and non-viable adult worms. In the group of worms selected as 'viable' around 70% kept their motility and metabolic activity at a high level until the end of the culture compared to the initial level. In contrast, none of the female worms and only 13% of the male worms categorized as 'poorly viable' demonstrated a motility index or metabolic level at the end of the culture period that was comparable to that of the worms in the 'viable' groups. For female worms the lactate output correlated significantly with weight whereas no correlation was seen between MTT reduction and weight.

Carbohydrate Catabolism in Adult <i>Onchocerca volvulus:</i> An Immunohistochemical Study

“…It has long been accepted that most filarial worms use anaerobic glycolytic breakdown of carbohydrates as their preferred route to supply energy for their requirements [26]. This has been the general opinion of most investigators of O. volvulus as well and this opinion is the basis for one of the most dependable viability test on O. volvulus during in-vitro drug assessments [27]. In this test the determination of the amount of lactate produced by a female worm within a stipulated period of incubation is used as the measurement of its level of activity.…”

Assessing the Viability and Degeneration of the Medically Important Filarial Nematodes

“…There are several studies that have described several enzymes that appear to be present in filarial worms [14], and it is likely that specific enzymes will be identified soon whose presence could act as reliable indicators of worm viability. Biochemical approaches have been used to assess the viability of in vitro worms for many years-the formazan assay being commonly used [15][16][17]. Enzymes involved in general biochemical maintenance of nematodes [18], such as Nras have already been seen to provide some information as to the adult worms' integrity after chemotherapy [7].…”

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

“…The viability of males was determined by assessment of the motility index of the whole worm as measured by a micromotility meter (Bennett and Pax 1986) at 4± 6 h postnodulectomy. Only males with a high motility index, indicating good viability (Strote et al 1993), were used for the morphology studies.…”

“…Reduction of MTT by living worms was determined spectrophotometrically at 510 nm. High values of absorption correlated with high viability of the worms (Strote et al 1993). Heat-killed worms were prepared by incubation of viable worms in medium at 56°C for at least 30 min.…”

“…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.…”