|WEEKS, JANICE - University Of Oregon|
|ROBERTS, WILLIAM - University Of Oregon|
|ROBINSON, KRISTIN - University Of Oregon|
|KEANEY, MELISSA - George Washington University|
|VERMEIRE, JON - University Of California|
|LOCKERY, SHAWN - University Of Oregon|
|HAWDON, JOHN - George Washington University|
Submitted to: International Journal for Parasitology: Drugs and Drug Resistance
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/17/2016
Publication Date: 12/1/2016
Citation: Weeks, J.C., Roberts, W.M., Robinson, K.J., Keaney, M.I., Vermeire, J.J., Urban Jr, J.F., Lockery, S.R., Hawdon, J.M. 2016. Microfluidic platform for electrophysiological recordings from host-stage hookworm Ascaris suum larvae: a new tool for anthelmenthic research. International Journal for Parasitology: Drug and Drug Resistance. 6:314-328. doi: 10.1016/j.ijpddr.2016.08.001.
Interpretive Summary: Current anthelmintic (anti-worm) drugs have limitations. Some parasites, like human and pig whipworms are relatively insensitive to all available anthelmintic drugs. In addition, the increasing development of drug resistance in parasites is weakening the effectiveness of current anthelmintic drugs, especially in veterinary medicine where drugs are failing in both livestock and companion animals. In human, reports of reduced anthelmintic efficacy are expected to increase. New anthelmintic treatments are urgently needed but the current drug development pipeline is inadequate to meet these needs. A previous study validated the use of an 8-channel microfluidic electro-pharyngeogram (EPG) platform in a free-living worm system, including characterization of the dose-dependent inhibition of pumping by several anthelmintic drugs and distinguishing wild type from drug-resistant worms. Because of the deleterious effect of parasitic nematode infections, however, there is a need to adapt this technology for use with parasitic nematodes that impact human and animal health. The present study optimized chip design and recording conditions for two classes of human and livestock soil-transmitted helminths (STHs); (1) Hookworms, including Ancylostoma ceylanicum, a significant human parasite in Southeast Asia; and (2) Ascaris suum, a zoonotic species for the human parasite, A. lumbricoides. These experiments demonstrated successful adaptation of the EPG platform for use with STH larvae, providing a new tool for anthelmintic research and investigations of nematode feeding behavior. These studies will be of interest to industry partners that screen for new anthelmintic drugs for use in veterinary and human medicine, and those that study worm physiology and mechanisms of action of biological mediators.
Technical Abstract: The screening of candidate compounds and natural products for anthelmintic activity is a key component of discovering new drugs against human and animal parasites. We previously validated in Caenorhabditis elegans a microfluidic device (‘chip’) that records non-invasively the tiny electrophysiological signals generated by rhythmic contraction (pumping) of the worm’s pharynx. These electropharyngeograms (EPGs) are recorded simultaneously from multiple worms per chip, providing a medium-throughput readout of muscular and neural activity, which is especially useful for compounds targeting neurotransmitter receptors and ion channels. Microfluidic technologies have transformed C. elegans research and the goal of the current study was to validate hookworm and Ascaris suum host-stage larvae for use with the microfluidic EPG platform. A. ceylanicum and A. caninum infective L3s (iL3s) activated in vitro generally produced erratic EPG activity under the conditions tested. In contrast, A. ceylanicum L4s recovered from hamsters exhibited robust, sustained EPG activity, consisting of three waveforms: (1) conventional pumps similar to those in other nematodes; (2) rapid voltage deflections, associated with irregular contractions of the esophagus and openings of the esophogeal-intestinal valve (a behavior we termed a ‘flutter’); and (3) hybrid waveforms, which we classified as pumps. For data analysis, pumps and flutters were combined and termed EPG ‘events’. EPG waveform identification and analysis were performed semi-automatically using custom-designed software. The neuromodulator serotonin (5-hydroxytryptamine; 5HT) increased EPG event frequency in A. ceylanicum L4s at an optimal concentration of 0.5 mM. The anthelmintic drug ivermectin (IVM) inhibited EPG activity in a concentration-dependent manner. EPGs from A. suum L3s recovered from pig lung exhibited robust pharyngeal pumping in 1 mM 5HT, which was inhibited by IVM. These experiments validate the use of A. ceylanicum L4s and A. suum L3s with the microfluidic EPG platform, providing a new tool for screening anthelmintic candidates or investigating parasitic nematode feeding behavior.