Progress 10/01/15 to 09/30/16
Outputs
Target Audience:We have written an abstract for the University of Georgia College of Veterinary Medicine annual report for 2016, describing the aims and progress to date of the project. This will be circulated to interested parties, including the Georgia State Legislature, DVM graduates of the Collges and others. Changes/Problems:Mr Storey, co-investigator on the project, developed an MRSA infection during surgery and was on extended sick leave for much of the time-period. This led us to place greater emphasis on the electrophysiological approaches rather than video microscopy. What opportunities for training and professional development has the project provided?Dr Wolstenholme and Mr Storey have learned about the Nemametrix equipment for electrophysiological recordings from nematode pharynxes. How have the results been disseminated to communities of interest?An abstract has been produced for the UGA CVM annual report. The research will be presented at the 2017 meeting of the American Association of Veterinary Parasitologists. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
. Feeding in nematodes involves coordinated and rhythmic contractions in the muscles of the throat, or pharynx. This process of pharyngeal pumping is necessary for the parasite to feed itself and is both an attractive target for the development of new antiparasitic drugs and the probable target of some of the existing ones. We have used video-microscopy, fluorescent dye ingestion and electrophysiological recordings to study the physiological and pharmacological properties of pharyngeal pumping in H. contortus, in the hope of optimizing an experimental platform for medium-throughput screening of potential anthelmintic compounds that impair the worms' health or feeding. Our studies focused on the fourth larval stage (L4) of the parasite life cycle. Though it is the adult worms that cause disease, these cannot be easily studied in the laboratory. The early larval stages (L1 to L3), though easy to cultivate and study, are less relevant for our purposes; the L3 stage does not feed at all, and the L1 and L2 stages eat bacteria rather blood. We therefore cultured L3 larvae, collected from the stool of infected animals and able to be stored in the refrigerator indefinitely, to the more active, host-stage L4 forms. We first confirmed that worms cultured to L4 in vitro exhibited pharyngeal pumping, as do L4 isolated from experimentally infected gerbils. Using video-microscopy at room temperature, we recorded pharyngeal pumping events in L4s taken from gerbils, and showed that pumping was stimulated by exogenous application of 5-hydroxytryptamine (5-HT, also known as serotonin), the neuromodulator that activates feeding in other nematode species. However, the rate of pumping was somewhat irregular and erratic. Dr. Janis Weeks and Kristin Robinson at University of Oregon investigated pharyngeal pumping in L4s cultured from L3s in vitro, using two methods: fluorescent dye ingestion and electrophysiology. A common method for evaluating feeding in cultured larvae is to incubate them with a fluorescent substrate, such as FITC-conjugated bovine serum albumin (BSA), to visualize ingestion of dye into the gut. The original fluor, FITC, proved problematic due to autofluorescence of the parasites, whereas Alex-Fluor 555-conjugated BSA worked well. Dye ingestion was most reliable in serum-augmented culture medium containing a low concentration of 5-HT. A more quantitative and reproducible approach for assaying feeding is to record the electrical signals emitted by pharyngeal muscles and neurons, termed an electropharyngeogram (EPG). A microfluidic device (chip) developed to record EPGs from other parasitic and non-parasitic nematodes in the Weeks lab was customized for use with the H. contortus L4; these long, thin larvae have an annoying tendency to coil up but with careful handling can be positioned in microfluidic recording channels. The chips and recording instrumentation were provided by NemaMetrix, Inc. (www.nemametrix.com), a spin-off company from the University of Oregon. The optimization of conditions that promote robust pumping--to permit screening for compounds that inhibit pumping--is an iterative, ongoing process. We have obtained EPG recordings from L4 at 37°C, but the pumping has been erratic compared to other nematodes, including human hookworm, that have been recorded in microfluidic chips. In hookworm, Weeks and collaborators found that whereas dye ingestion indicates successful activation of L3 to L4 in vitro, it does not predict which worms will produce robust, sustained EPG activity. It is not yet clear whether the feeding habit of H. contortus differs intrinsically from other species, or whether we have simply not identified optimal recording conditions. In summary, we are using three different techniques to study feeding in the L4 stage of H. contortus with the overall aims of learning more about the fundamental biology of this behavior and of producing an experimental platform for studying existing anthelmintic drugs and potentially seeking new ones.
Publications
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