Date of Award
Master of Science
Veterinary Microbiology and Preventive Medicine
Richard J. Martin
Parasitic nematode infections persist as a serious global public health threat to humans and animals. These infections cause debilitating conditions in humans and significant economic losses through infection of livestock and crop damage. One of the many parasitic nematode infections is lymphatic filariasis; one of the causative agents is Brugia malayi. Presently, the treatment of parasitic infection relies on anthelmintics as there are no vaccines. Therefore, regular use of the same drugs is expected to produce either loss of potency or resistance. Resistance to anthelmintics compromises the control of nematode infections and is also a major problem in veterinary and human medicine, resulting in untold morbidity and even mortality. Therefore, deciphering pharmacological targets in parasitic nematode species is an urgent need to identify potential molecular mechanisms associated with resistance and drug discovery. nAChRs (nicotinic acetylcholine receptors) will continue to present targets for such strategies. nAChRs are homo- or hetero-pentameric ligand-gated ion channels mediating excitatory neurotransmission and muscle activation. In Brugia, there are multiple subtypes of nicotinic receptors on the body muscle (M-, P-, L, and N-) that are sensitive to different cholinergic anthelmintics. Levamisole is an anthelmintic drug that acts by activating an L-type of nAChR at the nematode neuromuscular junction to cause paralysis.
We hypothesized that the populations of receptor subtypes are dynamic and change to compensate and limit the effects of anthelmintic exposure. Therefore, we investigated the issues of anthelmintic resistance in vitro using Worminator, which quantitatively measures the motility of B. malayi and qRT-PCR (quantitative real time polymerase chain reaction) to analysis the relative expression changes of nAChR subunit mRNAs. These were performed to characterize the phenotypic and molecular analysis of nAChR gene expression at different concentrations and time intervals.
We measured the in vitro effects of levamisole on the motility of adult male and female Brugia malayi at different time points. We also determined real time IC50 values of different concentrations of levamisole at 10 minutes against adult B. malayi which was 10 nM. Both, high and low drug concentrations induced an immediate spastic paralysis that lasted for up to one hour in worms. However, female worms completely recovered around 4 hours later, whereas male worms were only partially desensitized. This suggests a difference in sensitivity to levamisole between male and female worms. The variation in motility during levamisole incubation was possibly due to differences in nAChR genes expression. Completely recovered female worms had upregulation of the Bma-unc-38 gene (ANOVA, P < 0.01). Partially desensitized male worms also showed significant upregulation of Bma-unc-38 (ANOVA, P < 0.01). This indicates that the Bma-unc-38 gene upregulation plays a key role for motility in male and female worms. However, the motility variation may be possibly due to differences in nAChR genes expression of other nAChR genes also. Female worms under flaccid paralysis had increased Bma-unc-29 (ANOVA, P < 0.01).
Therefore, we concluded that both Bma-unc-38 and Bma-unc-29 genes are important for the pharmacology of L-type receptors and motility. Together expression of these genes may induce a functional receptor which responded to levamisole. In male worms, robust increased expression of Bma-unc-38 and Bma-unc-29 together might delay desensitization. However, in female worms, robust expression of Bma-acr-8 and Bma-unc-38 may result in failure to form a functional receptor to respond to levamisole.
Mengisteab T Wolday
Wolday, Mengisteab T., "Phenotypic and molecular analysis of desensitization to levamisole in male and female adult Brugia malayi" (2019). Graduate Theses and Dissertations. 17809.