MOLECULAR PHARMACOLOGY OF RESISTANCE TO CHOLINERGIC ANTHELMINTICS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: ANIMAL HEALTH
• Reporting Frequency: Annual
• Accession No.: 0221578
• Project Start Date: Oct 1, 2009
• Project End Date: Sep 30, 2012
• Program Code: [(N/A)]- (N/A)

Recipient Organization
IOWA STATE UNIVERSITY
2229 Lincoln Way
AMES,IA 50011

Performing Department
Veterinary Medicine

Non Technical Summary
Parasitic nematodes such as Oesophagostomum dentatum have major impact on human and animal health in the US and globally. Unfortunately, resistance of this and other parasitic nematodes to antihelmintic drugs including levamisole have been reported. This resistance is predicted to involve the levamisole receptor and its 5 subunit proteins. However, the mechanism by which resistance is achieved has not been demonstrated. One likely mechanism is that mutations within receptor subunits within resistant worms causes the resistance. If so, then the gene sequences for receptor subunits should be different in sensitive versus resistant worms. Another possibility is that differences in which subunits assemble to form the receptor results in sensitive or resistant receptors. One outcome of this study will be a determination of receptor subunit gene sequences from resistant and sensitive worms, and an analysis of those sequences for changes that predominate only in genes from resistant worms. Another outcome will be testing whether recombinant receptors derived from resistant or sensitive worms and expressed in frog cells exhibit different levamisole resistance phenotypes.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
313	3130	1080	10%
313	3130	1110	10%
313	3130	1120	20%
311	3130	1040	10%
311	3130	1080	10%
311	3130	1110	20%
311	3130	1120	10%
313	3130	1040	10%

Knowledge Area
311 - Animal Diseases; 313 - Internal Parasites in Animals;

Subject Of Investigation
3130 - Nematodes;

Field Of Science
1040 - Molecular biology; 1120 - Nematology; 1110 - Parasitology; 1080 - Genetics;

Keywords

nematode

drug resistance

levamisole resistance

oesophagostomum dentatum

Goals / Objectives
Parasitic nematodes such as Oesophagostomum dentatum have major impact on human and animal health in the US and globally. While resistance of this and other parasitic nematodes to antihelmintic drugs including levamisole have been reported, and is predicted to involve the levamisole receptor and its 5 subunit proteins, the mechanism by which resistance is achieved is unknown. The goals of this study are (i) to determine whether levamisole receptor protein sequences vary among levamisole-resistant and levamisole-sensitive nematodes, and (ii) express recombinant receptor subunits for assessment of levamisole-associated electrophysical properties of the receptor. Expected outputs will include conducting and analyzing the experiments among the collaborating research groups, training and mentorship of graduate and advanced trainees in the area of parasitology and bioinformatics, and dissemination and discussion of results via publications and national meetings of parasitologists.

Project Methods
Levamisole receptor protein subunits cDNAs will be PCR-cloned from levamisole-resistant and levamisole-sensitive populations of O. dentatum, each population isolated from a single infected animal. Cloning will use degenerate PCR-primers designed against genes that encode the homologous subunits in related nematode species. Sequence alignments will use standard software (e.g. Clustal). Data will be analyzed for nucleotide differences that group to either the resistant or the sensitive populations; such differences would indicate changes that may be causal to the sensitivity phenotype. Levamisole receptor subunits will be expressed in frog oocytes then record whole-cell currents will be taken in response to different concentrations of selective nicotinic anthelmintics.

Progress 10/01/09 to 09/30/12

Outputs
OUTPUTS: The main activities that were generated during this study were (i), conducting experiments in which gene expression was assessed in drug-resistant and -sensitive animal-pathogenic nematodes, with focus upon proteins likely to be involved in drug resistance, and (ii), training and mentoring of one PhD-candidate student and one research associate in techniques required for conducting and analyzing the experiments and in the background biological information associated with the study. Products generated and disseminated from this study included (i) one agricultural sciences PhD student whose graduate thesis focused upon experiments conducted within this study (PhD: College of Agriculture and Life Sciences: Interdepartmental Major in Genetics), (ii) collaboration initiated between the two research groups that conducted the experiments, (iii) genetic details of 26 genes/proteins that art predicted to be involved in drug resistance; these genetic details have been deposited on-line at the U.S National Center for Biotechnology Information so they can be available to the scientific community. PARTICIPANTS: The participants on this grant included the project directors Jeffrey Beetham, Richard Martin, and Alan Robertson who designed the experiments and mentored execution and analysis of the experiments by trainees. Trainees who performed the experimental execution on the project included PhD-student Nathan Romine and support scientist Christian Bartholomay. Primary analysis of the data was by Romine, Beetham, and Martin. TARGET AUDIENCES: The target audience that was served by the project is comprised primarily of scientists working in the field of drug resistance in nematode-pathogens of agricultural animals. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The findings of this study resulted in a change of knowledge comprised by the elucidation of genes that are implicated to be involved in nematode resistance to drugs. There was also a major change in knowledge for the trainees and scientists in regards to skills learned and developed with which to apply new bioinformatics techniques towards the identification and characterization of drug-resistance genes. Based upon the change in knowledge about the genes, and about the techniques of bioinformatics, a change of action impact of the study is that it is shaping the approach now being followed towards further study of the biological process of drug resistance in the parasitic nematodes.

Publications

• Romine, N. (2012). Transcriptomic identification and characterization of levamisole resistance associated genes in the swine nodular worm Oesophagostomum dentatum. Graduate Thesis and Dissertation. http://lib.dr.iastate.edu/etd Romine, N., Martin, R., Beetham, J. (2013) Computational cloning of drug target genes of a non-model nematode, Oesophagostomum dentatum. BMC Genetics (pending).