In Vitvo Studies on the Relative Sensitivity to Ivermectin of Necator americanus and Ancylostoma ceylanicum

-Richards J. C., Rebnke J. M. & Duce I. R. 1995. In uifro studies oo tbe relative sensitivity to ivermectin of Necator americanus and AncyIostoma ceyhnicum. International Journal for Parasitology 25: 1185-1191. Experiments were carried out to compare tbe sensitivity of Ancylostoma ceyhnicum and Necator umericunus to ivermectin (IVM) and pyrantel in vitro. Loss of motility and inhibition of ingestion by IVM were compared and A. ceyhicum was found to be approximately 40-50 times more sensitive to IVM than N. umericunus. Roth species showed a similar sensithity to pyrantel. Uptake of 13HJIVM across tbe cuticle was compared and shown to bc unlikely to account for tbe differences in sensitivity to IVM between tbe two SpCieS.


INTRODUCTION
There is much interest in the potential of ivermectin (IVM) as a microfilaricide for the treatment of onchocerciasis (Bennett, Williams & Dave, 1988), and mass trials against onchocerciasis are being implemented in 12 African and Central American countries.IVM has proven to be a very effective antiparasitic drug and many of the common gastrointestinal nematodes are highly susceptible to IVM at concentrations much lower than those at which other anthelmintics are effective.However, some parasites, including Heligmosomoides polygyrus, Trichuris trichiura, and hookworms (Wahid, Behnke & Conway, 1989;Whitworth, Morgan, Maude, McNicholas & Taylor, 1991) have been found to be tolerant to IVM.
indicated that in a hamster model, Necator was relatively insensitive to IVM.
The difference in IVM sensitivity is of interest in two respects.First, the drug may be used clinically in situations where N. americanus and Ancylostoma duodenale are both present in the population and its use may influence epidemiology.
Second, if the differential sensitivity of these 2 species reflects a difference in the drug target site, these species of hookworm may provide a good model system to probe further the molecular action of the drug.
In this paper we report in vitro experiments which show that the difference in sensitivity of N. americanus and A. ceylanicum to IVM can be demonstrated in vitro and is therefore not attributable to the host-parasite relationship.We further show that the differential responsiveness cannot be explained by differences in the uptake of the drug.

MATERIALS AND METHODS
Parasites and hosts.As the [3H]IVM was found to be adsorbed by the wire ligatures, the ends (0.5 mm) of each worm (both ligatured and unligatured) were removed.
After being left to dry overnight on pieces of foil, individual worms were placed in scintillation vials.The worms were solubilized in 100 ul of 2.5 M NaOH for l-2 h.HCl, 100 u12.5 M, was then added, followed by 4 ml of scintillation fluid (Packard  For all a priori hypotheses examined, the test statistic z is given as appropriate.Probabilities (P) of 0.05 or less were considered significant.

RESULTS
Comparison of the effects of IVM and pyrantef on the activity of A. ceylanicum and N. americanus Over a period of 3 h, A. ceylanicum was found to be equally sensitive to IVM and IVM-PO4 and was immobilized by IVM or IVM-PO4 dose-dependently with an approximate ~~~~ at 3 h of 1.14 PM, 2.Even after incubation periods of 24-48 h, N. americanus was not visibly affected by IVM-PO4 at concentrations of 11.44 PM compared with controls incubated in normal media.However, this concentration caused >90% inactivity in A. ceylanicum after 3 h.
Pyrantel was found to be similarly effective at paralysing A. ceylanicum and N. americanus dosedependently, with approximate EQ~S of 1.34 and 3.5 PM (A. ceylanicum and N. americanus, respectively) (t = 3 h) (Fig. 2).

Uptake of [3H]ZVM by A. ceylanicum and N. americanus
There was no significant effect of ligaturing the worms on the uptake of IVM; however, there was a significant difference between the species with A. ceylanicum taking up more [3H]IVM than N. americanus (Table la).There was no difference in the uptake by male and female worms.

Uptake of [3H]inulin by A. ceylanicum and N. americanus
The ligatures had a significant effect on the uptake of [3H]inulin by both A. ceylanicum and N. americunus (ratio non-ligatured vs. ligatured 7 : 1, 9 : 1, respectively, Table lb), proving the effectiveness of the ligatures in preventing the entry of media via anatomical openings (Ho, Geary, Barshun, Sims & Thompson, 1992).N. americanus was more active in  terms of uptake, and ingested approximately 6 times more [3H]inulin than A. ceylanicum.In the medium used, the basal level of ingestion of [3H]inulin was lower in A. ceylanicum than N. americanus, suggesting that N. americanus is more active in terms of oral uptake under these conditions.
Comparison of the effect of IVM-PO4 on the uptake of [3H/inulin by A. ceylanicum and N. americanus Although N. americanus fed more actively than A. ceylanicum over a period of 24 h (RPM1 & HLac medium, Tables 2a, b) a significant inhibition of oral uptake of [3H]inulin by IVM-PO4 was demonstrated for both species (Tables 2a, b).
There was a highly significant difference between the two species with inulin uptake by A. ceylanicum being inhibited by much lower concentrations of IVM-PO4 than N. americanus (approximate dependently (Tables 2c, d, Fig. 3), with both species showing similar sensitivity (EC&S approximately 0.6 and 0.4 PM, N. americanus and A. ceylanicum respectively).

DISCUSSION
The results reported in this paper provide the first direct in vitro comparisons of the sensitivities of two species of hookworm from the important genera Ancylostoma and Necator to WM.Previous studies (Behnke et al., 1993) showed that in the hamster host, A. ceylanicum was on average 300 times more sensitive to IVM than N. americanus.Studies by Rajasekeriah et al. (1989) also reported the relative insensitivity of N. americanus to IVM in the hamster model.The results of the above in vitro experiments in which the effects of IVM and pyrantel on motility were assessed support the results of these previous in vivo experiments (Behnke et al., 1993;Rajasekeriah et al., 1989) as A. ceylanicum and N. americanus were shown to have similar sensitivities to pyrantel, whereas A. ceylanicum was found to be approximately 50 times more sensitive to IVM (t = 3 h) than N. americanus.The difference in responsiveness to IVM does not appear to be related to response time as concentrations of IVM-PO4 that immobilized A.
of hookworms to ivermectin in vitro 1189  ceylunicum in 3 h (1.14 pm, 50% worms immobilized) had no detectable effect on N. americanus over 24 h.
The experiments reported here used concentrations 2 1 PM, which have been previously criticized as being unrepresentatively high compared to effective in vivo concentrations (Geary, Klein, Vanover, Bowman & Thompson, 1992;Geary, Sims, Thomas, Vanover, Davis, Winterrowd, Klein, Ho & Thompson, 1993).The difference in sensitivity between the two species in vitro is also relatively lower than the difference in sensitivity shown in vivo (A.ceylanicum 4&50 times more sensitive than N. americanus in vitro, compared to a 20s300-fold difference in vivo).When comparing these in vitro experiments with experiments carried out in vivo, it is important to take into account a number of factors.For example, the in vitro experiments required a dose of IVM that had an acute physiological effect, resulting in a single endpoint (e.g.loss of motility) being demonstrated within the limited timescale of a few hours.In comparison, in vivo it is likely that a given drug affects worm viability by inhibiting or interfering with a range of processes in the parasite, e.g.inhibition of egg production, feeding, or movement.In vivo experiments with IVM also tend to involve much longer periods of exposure to the drug.For example, Campbell & Benz (1984) found that the anthelmintic efficacy of IVM in cattle persisted for 2 weeks after treatment.Similarly, the microfilaricidal activity of IVM was shown to persist in rodents for at least 30 days after a single dose (Zahner, Sanger, Lammler & Muller, 1987).
Using a micromotility meter, Geary et al. (1993) demonstrated that the motility of Haemonchus contortus was affected by concentrations of IVM 2 lo-* M, much lower than the concentrations of IVM at which loss of motility could be determined by observation (lo@ M).It is likely that IVM affects motility in A. ceylanicum and possibly N. americanus at much lower concentrations than those at which the end-point was determined in these experiments.Nevertheless, in these acute in vitro experiments both motility and ingestion showed a significantly greater sensitivity to IVM in A. ceylanicum than in N. americanus.
The cuticle of nematodes can act as an important permeability barrier and it is possible that the differences in sensitivity to IVM between the two Fig. 1.Comparison of the effects of IVM (Fig. la) and IVM-PO4 (Fig. lb) on % active A. ceylanicum and N. americanus.Worms were classed as either active or inactive (see Methods) and the data are presented as the mean percentage ( f S.E.M.) of worms showing activity after 1, 2 and 3 h incubation in IVM or IVM-PO4 (n = 30-60 worms).IVM-PO4 was shown to immobilize N. americanus dose-dependently (Spearman rank rs -0.898, n = 18, P<O.OOOl, t = 3 h).Similarly IVM and IVM-PO4 both caused inactivity in A. ceylanicum dose-dependently (Spearman rank r, -0.80, n = 55, P<O.OOOl, t = 3 h; T, -0.986, n = 13, P<O.OOOl, t = 3 h, respectively).

Table la -
The uptake of [3H]IVM by A. ceylunicum, N.