Development of an In Vivo Exsheathment Assay of Infective L3 Haemonchus contortus Larvae in Fistualated Sheep

Author(s)

Holly N. Williams, University of Rhode IslandFollow
Katherine Petersson, University of Rhode IslandFollow
Carly Barone, University of Rhode IslandFollow

Major

Animal Science and Technology

Advisor

Petersson, Katherine

Advisor Department

Animal and Veterinary Science

Advisor

Barone, Carly

Advisor Department

Animal and Veterinary Science

Date

5-2015

Keywords

Small ruminant; Internal parasite; Exsheathment; Fistula; Assay; Larva

Creative Commons License

This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 3.0 License.

Abstract

Purpose: Gastrointestinal nematodes (GIN) hinder the sustainable production of small ruminants on pasture and parasite resistance to chemical dewormers is becoming a growing concern. Condensed tannin containing legume forages are being tested to evaluate their anti-parasitic properties and potential contribution to an overall parasite control program for small ruminants such as sheep and goats. One of the most pathogenic GIN of small ruminants is Haemonchus contortus. The final step to full infectivity of H. contortus third stage larvae (L3) is exsheathment in the rumen. The objective of this study was to establish an in-vivo exsheathment assay in fistulated sheep as a tool to evaluate the ability of several varieties of birdsfoot trefoil, a condensed tannin containing forage, to inhibit the exsheathment of H. contortus L3. Methods: The larvae used in the experiment was harvested from the feces of infected donor lambs using a modified Baermann’s technique. In addition, two in-vitro exsheathment assays using either dilute bleach solution or overnight incubation after exposure to CO₂ were used for comparison against in-vivo exsheathment. Three different methods of containment of the infective L3 in a porous PVC tube within the rumen were evaluated. 1) Histology cassettes fitted with nylon mesh with a pore size of 8 microns. 3000 larvae were pipetted into the cassettes and the cassettes sealed. To prevent the escape of L3 various methods of sealing the cassettes were tried including super glue, clamps, rubber bands, and clay. 2) Dialysis tubes. 2000 larvae were pipetted into dialysis tubing with varying molecular weight cut offs (50 to 1,000 kD). 3) Small (5 ml) polypropylene sample jars with 8 micron mesh cover. 2000 L3 pipetted into polypropylene sample jars covered with an 8 micron mesh. Each method was tested by suspending the larvae in the various containment systems within a porous PVC tube and then removing a container after 60 and 120 minutes of incubation within the rumen. The percentage of viable and exsheathed infective L3 larvae were determined by microscope. Criteria used to evaluate the efficacy of containment were the percent of recovered L3 from the containment system and the percent of total L3 exsheathed. Results: Results from the histology cassette technique show that, although some L3 were exsheathed, the histology cassettes were not able to be sealed to the degree necessary to prevent L3 from escaping, thus potentially infecting the sheep. Results are still pending on the other two methods as we continue to collect data. Conclusion: Development of an in-vivo exsheathment assay is critical in determining the anthelmintic efficacy of condensed tannin containing legume forages. In order to see if the forages affect exsheathment it is necessary to have an in-vivo assay that is able to provide reliable control-exsheathment from which you can compare exsheathment statistics after being fed the forages.