Georgia Veterinary Scholars Program at the UGA College of Veterinary Medicine

The University of Georgia College of Veterinary Medicine

2005 Georgia Veterinary Scholars

Georgia Veterinary Scholar	Faculty Mentor

Cory Gresham University of Georgia Class of 2008	Dr. Ray Kaplan

Cloning and Characterization of the Glutamate-Gated Chloride Channel Gene of Parascaris equorum

Cory S. Gresham 1*, Ray M. Kaplan 2

1 Georgia Veterinary Scholars Program, University of Georgia, College of Veterinary Medicine. 2Department of Infectious Diseases, University of Georgia, College of Veterinary Medicine

Parascaris equorum infection is the most common and important parasitic pathogenof foals, resulting in pulmonary and gastrointestinal dysfunction, poor growth, general ill-thrift and possibly death. Ivermectin/moxidectin are the most popular drugs used to control P. equorum infection; in some cases foals are treated as frequently as every 30 days. Ivermectin and moxidectin belong the avermectin/milbemycin class of anthelmintics, which target the glutamate-gated chloride channel (GluCl) of nematodes. Recent reports of apparent ivermectin resistance from Canada and ivermectin/moxidectin resistance from the Netherlands indicate drug resistant P. equorum may be reaching a problematic level. Molecular analysis of this important drug target may improve understanding of the molecular basis of avermectin/milbemycin resistance in this parasite. Adult P. equorum samples were collected at necropsy from foals of a herd that had never been treated with anthelmintics. RNA and DNA extractions were performed using a CTAB/chloroform protocol. Degenerate primers were synthesized based on an alignment of a highly conserved region of the GluCl gene from other nematodes (Caenorhabditis elegans, Cylicocyclus nassatus, and Haemonchus contortus). The C. nassatus sequence predicts a 1kb fragment. Polymerase chain reactions produced a fragment of approximately 1kb as well as 700, 600, 400, 300 and 200 bp fragments. The 1000 bp amplicon was isolated and ligated into a plasmid vector and transformed into Escherichia coli. Blue/white screening followed by electrophoresis confirmed the presence of the cloned fragment, and several different plasmid preparations were submitted for sequencing. The returned sequence was compared to previously sequenced GluCl channel genes of other nematodes.