Invert Neurosci (2015) 15:5 DOI 10.1007/s10158-015-0181-0


Transient effects of levamisole on Brugia malayi microfilariae Eman Mostafa1,2,3 • Bob Storey1 • Adel Mohammed Farghaly3 • Hany Abd El-Hai Afify3 • Afaf Abd El-Raouf Taha3 • Adrian J. Wolstenholme1,2

Received: 1 April 2015 / Accepted: 9 July 2015 / Published online: 18 July 2015 Ó Springer-Verlag Berlin Heidelberg 2015

Abstract Levamisole is an anthelmintic drug that acts by activating nicotinic acetylcholine receptors at the nematode neuromuscular junction and causing paralysis. We measured the in vitro effects of levamisole on the motility of Brugia malayi microfilariae; after 2 h incubation the apparent IC50 was 2.68 mM. Lower drug concentrations, such as 1 mM, caused an immediate total paralysis that lasted for up to 1 h, but was completely reversed by 2 h of incubation. The ‘recovered’ parasites were still completely susceptible to application of a second nicotinic agonist, pyrantel. Keywords Nicotinic acetylcholine receptor  Nematode  Anthelmintic  Brugia malayi Levamisole is an agonist at nicotinic acetylcholine receptors (nAChRs) found at the nematode neuromuscular junction (Martin 1997). It and other nicotinic agonists are widely used as anthelmintic drugs against gastrointestinal parasites of livestock, but have not been widely used against filarial parasites which inhabit tissues other than the GI tract. Recently, it has been shown that adult Brugia malayi, the causative agent of about 10 % of cases of lymphatic filariasis, possesses levamisole-sensitive nAChRs (Robertson et al. 2011, 2013). We therefore & Adrian J. Wolstenholme [email protected] 1

Department of Infectious Diseases, University of Georgia, 501 D.W. Brooks Drive, Athens, GA 30602, USA


Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA, USA


Department of Parasitology, Zagazig University, Zagazig, Egypt

undertook to study the effect of the drug on the locomotion of microfilariae (Mf). There have been several recent descriptions of improved systems for the measurement of nematode movement, and we used the recently described Worminator system for microscopic life-stages of nematodes in these experiments (Storey et al. 2014). Brugia malayi Mf isolated from the peritoneal cavity of infected Mongolian gerbils were provided by the Filarial Research Reagent Resource Center (FR3: Athens, GA, USA). The Mf were purified by centrifugation through 5 ml of HistopaqueTM (Sigma, St. Louis, MO, USA) for 5 min at 2000 rpm in an Allegra X-30 centrifuge and washed twice in PBS before being re-suspended in 10mls RPMI-1640 (Life Technologies, Grand Island, NY, USA). A total of 10 mM stocks of levamisole hydrochloride (Sigma) were freshly prepared in deionized water and diluted in RPMI-1640 to provide working stocks ranging from 2 nM to 5 mM. Stocks of pyrantel tartrate (Sigma) were prepared in DMSO and diluted in RPMI-1640 so that the final concentration of DMSO was less than 1 % (v/v). The motility assays were carried out on the Worminator system in black 384-well tissue-culture plates with optically clear bottoms (Thermo Scientific Nunc #142761) as previously described (Storey et al. 2014), except that the time-lapse option of WormAssay v1.4.1 was used. The data were collected by WormAssay (Marcellino et al. 2012) and analyzed using Graphpad Prism v6.05. All experiments were carried out in triplicate with three biological replicates. When B. malayi Mf were incubated in various concentrations of levamisole at 37 °C for 2 h and the effect of the drug on their motility assessed, a dose–response curve with an apparent IC50 of 2.68 mM (95 % CI 2.37–3.02 mM) was generated (Fig. 1a). However, visual examination of the Mf indicated that they were immediately paralyzed by


5 Page 2 of 3

Invert Neurosci (2015) 15:5

Fig. 1 In vitro effects of levamisole on the motility of B. malayi Mf. a Dose–response curve following 2 h incubation. b Time-course of the motility of Mf following exposure to various concentrations of levamisole

reasons for the transient paralysis seen in 1 mM levamisole might be that this concentration of drug causes the nAChRs at the neuromuscular junction to be downregulated or otherwise desensitized (Taly et al. 2009). Agonist-induced endocytosis of mammalian neuromuscular receptors has been observed within 5 min of drug application (John and Gordon 2001). If this was the cause of the effects observed here, we reasoned that Mf pretreated with 1 mM levamisole that had recovered from paralysis should also be more resistant to the effects of a second nicotinic agonist, such as pyrantel. However, these worms were still as completely paralyzed by 150 lM pyrantel (Storey et al. 2014) as untreated parasites (Table 1), suggesting that at least some of the nicotinic receptors are still active following levamisole exposure. Of course, this interpretation relies on levamisole and pyrantel acting on the same targets. Though this seems likely, and there is evidence for this from other parasitic nematodes, such as Ascaris suum (Williamson et al. 2009), it has not been formally proven for B. malayi. There are likely to be multiple nAChR subtypes on B. malayi muscle cells, as reported for many other nematodes (Qian et al. 2006; Bennett et al. 2012; Jones and Sattelle 2004; Touroutine et al. 2005; Williamson et al. 2007; Li

lower drug concentrations, but had recovered by the time the readings were taken. We therefore used the time-lapse option of WormAssay to measure the motility of the Mf every 5 min for 30–40 min after addition of the drug. Figure 1b shows that 0.1 mM levamisole had little effect on Mf motility at any time, but that 1 mM caused an immediate paralysis that lasted for 1 h, and this was relieved by 2 h incubation. Addition of 5 mM levamisole caused a paralysis that did not reverse by 2 h (Fig. 1b) or 24 h (motility \1 mmu). We did not observe a similar phenomenon with any other nicotinic anthelmintic, including pyrantel, morantel, oxantel, and tribendimidine (Storey et al. 2014). Table 1 shows that the motility of Mf incubated in 1 mM levamisole was significantly different from the water control after 5 min (p = 0.008), but not after 2 h (p = 0.73). In contrast, the motility of Mf incubated in 150 lM pyrantel was significantly reduced from the 1 % (v/v) DMSO control after both 5 min (p = 0.044) and 2 h (p = 0.045) incubation. The results presented here are similar to those reported for adult female B. malayi, where 30 lM levamisole inhibited motility by 85 % in 10 min, but the worms recovered within an hour (Robertson et al. 2013). Possible

Table 1 Motility of B. malayi Mf after 1 mM levamisole treatment Treatment 1

Motility 5 min post-treatment 1

Motility 2 h post-treatment 1

Treatment 2

10 % Water

12.71 ± 4.1

26.81 ± 12.8


1 % DMSO 1 mM levamisole

18.04 ± 6.82 1.29 ± 1.35

28.43 ± 15.15 21.01 ± 3.3

None 1 % DMSO

1 mM levamisole 150 lM pyrantel


1.29 ± 1.35


0.08 ± 0.12


150 lM pyrantel

21.01 ± 3.3


0.017 ± 0.005

Motility 5 min post-treatment 2

17.76 ± 6.52d 0.16 ± 0.12c,d


Parasites were incubated in 1 mM levamisole for 2 h and then exposed to 150 lM pyrantel or vehicle for 5 min. Their motility was measured using the Worminator system, and values are given in mean motility units (mmu). Values are shown ± standard deviation a

p value for the effects of 1 mM levamisole and 150 lM pyrantel 5 min post-treatment = 0.18


2 h post-treatment, p value = 0.006


Comparison of the effects of 150 lM pyrantel alone and after 2 h of exposure to 1 mM levamisole, p = 0.41


Comparison of the effects of 1 % DMSO and 150 lM pyrantel after 2 h exposure to 1 mM levamisole, p = 0.0012 (all comparisons using unpaired t test with Welch’s correction)


Invert Neurosci (2015) 15:5

et al. 2015), and it is possible that pyrantel acts on additional receptors that may not be sensitive to levamisole, or that the binding sites do not overlap. Pyrantel has channelblocking activity (Harrow and Gration 1985), and this may be responsible for at least some of its paralytic effects on B. malayi Mf. It is also possible that drug extrusion mechanisms or other detoxification processes may be involved, though little is known about these in nematodes with regard to levamisole. Levamisole does have activity against B. malayi Mf in vivo, and lower doses of the drug (25 mg/kg, roughly equivalent to 0.12 mM assuming the drug is evenly distributed throughout the animal) have been reported to have a longer-lasting and stronger effect of Mf levels in infected Mastomys coucha than higher concentrations (50 mg/kg) (Zahner and Schares 1993). This phenomenon was not seen in animals infected with other filarial nematodes, including B. pahangi. Whether or not the transient paralysis we report here is relevant to this observation, our data and those of Robertson et al. (2013) do indicate that the interactions of nicotinic agonists with their target receptors in B. malayi are complex, as in other parasitic nematodes. Acknowledgments This work was supported by funds from the Egyptian Cultural and Educational Bureau, Washington, DC, USA. We thank Dr Barbara Reaves for a critical reading of the manuscript. Compliance with ethical standards Conflict of interest


References Bennett HM, Williamson SM, Walsh TK, Woods DJ, Wolstenholme AJ (2012) ACR-26: a novel nicotinic receptor subunit of parasitic nematodes. Mol Biochem Parasitol 183:151–157 Harrow ID, Gration AF (1985) Mode of action of the anthelmintics morantel, pyrantel and levamisole on muscle cell membrane of the nematode Ascaris suum. Pestic Sci 16:662–672 John PAS, Gordon H (2001) Agonists cause endocytosis of nicotinic acetylcholine receptors on cultured myotubes. J Neurobiol 49:212–223

Page 3 of 3 5 Jones AK, Sattelle DB (2004) Functional genomics of the nicotinic acetylcholine receptor gene family of the nematode, Caenorhabditis elegans. BioEssays 26:39–49 Li B-W, Rush AC, Weil GJ (2015) Expression of five acetylcholine receptor subunit genes in Brugia malayi adult worms. Int J Parasitol Drugs Drug Resist 5:100–109 Marcellino C, Gut J, Lim KC, Singh R, McKerrow J, Sakanari J (2012) WormAssay: a novel computer application for wholeplate motion-based screening of macroscopic parasites. PLoS Negl Trop Dis 6:e1494 Martin RJ (1997) Modes of action of anthelmintic drugs. Vet J 154:11–34 Qian H, Martin RJ, Robertson AP (2006) Pharmacology of N-, L-, and B-subtypes of nematode nAChR resolved at the singlechannel level in Ascaris suum. FASEB J 20:E2108–E2116 Robertson AP, Puttachary S, Martin RJ (2011) Single-channel recording from adult Brugia malayi. Invertebr Neurosci 11:53–57 Robertson AP, Buxton SK, Martin RJ (2013) Whole-cell patch-clamp recording of nicotinic acetylcholine receptors in adult Brugia malayi muscle. Parasitol Int 62:616–618 Storey B, Marcellino C, Miller M, Maclean M, Mostafa E, Howell S et al (2014) Utilization of computer processed high definition video imaging for measuring motility of microscopic nematode stages on a quantitative scale: ‘‘The Worminator’’. Int J Parasitol Drugs Drug Resist 4:233–243 Taly A, Corringer P-J, Guedin D, Lestage P, Changeux J-P (2009) Nicotinic receptors: allosteric transitions and therapeutic targets in the nervous system. Nat Rev Drug Discov 8:733–750 Touroutine D, Fox RM, Von Stetina SE, Burdina A, Miller DM, Richmond JE (2005) acr-16 encodes an essential subunit of the levamisole-resistant nicotinic receptor at the Caenorhabditis elegans neuromuscular junction. J Biol Chem 280:27013–27021 Williamson SM, Walsh TK, Wolstenholme AJ (2007) The cys-loop ligand-gated ion channel gene family of Brugia malayi and Trichinella spiralis: a comparison with Caenorhabditis elegans. Invertebr Neurosci 7:219–226 Williamson SM, Robertson AP, Brown L, Williams T, Woods DJ, Martin RJ et al (2009) The nicotinic acetylcholine receptors of the parasitic nematode Ascaris suum: formation of two distinct drug targets by varying the relative expression levels of two subunits. PLoS Pathog 5:e1000517 Zahner H, Schares G (1993) Experimental chemotherapy of filariasis—comparative-evaluation of the efficacy of filaricidal compounds in Mastomys coucha infected with Litomosoides carinii, Acanthocheilonema viteae, Brugia malayi and Brugia pahangi. Acta Trop 52:221–266


Transient effects of levamisole on Brugia malayi microfilariae.

Levamisole is an anthelmintic drug that acts by activating nicotinic acetylcholine receptors at the nematode neuromuscular junction and causing paraly...
446KB Sizes 1 Downloads 12 Views