Effect of Wine Flour Containing Condensed Tannin on Development of Coccidian Oocysts and Parasitic Nematode Larvae on Small Ruminants

Graduate #27
Discipline: Biological Sciences
Subcategory: Microbiology/Immunology/Virology

Michelle M. Opiri - Louisiana State University
Co-Author(s): James E. Miller, D.V.M., Ph.D., & Vicky E. Kelly, Ph.D. Candidate.

Internal parasites are a major problem for the US poultry and small ruminant industries, loses annually exceed $1.5 billion due to poultry coccidiosis. Coccidiosis is caused by the apicomplexan protozoa Eimeria that infects intestinal cells of susceptible hosts, severely impairing growth and feed utilization. Similarly, gastrointestinal nematode(GIN) parasites are also found in the GI tract and affect growth and feed utilization. Both parasites have been historically controlled by medication however due to increasing levels of drug-resistant strains, costs of new drugs and the mistrust of drug-treated meat products, worldwide calls for the development of alternative control methods have occurred. Therefore, condensed tannin (CT) containing plants are being evaluated as an alternative form of control. CTs are polyphenolic compounds, found in the seeds and foliage of some legumes and grass cultivars. Wine flour (WF), which contains CT in grape skins (procyanidins and prodelphinidins) and seeds (procyanidins only), is a by-product of making wine. This study was conducted to evaluate the effect of WF on excystation of Eimeria spp. Oocysts and growth and development of GI nematode larvae in feces. Oocyst Excystation Assay was conducted here 2%, 0.2%, 0.02%, and 0.002% solutions were made of Cab Franc, Cab Sauv, Amprolium(Corid), and a negative control of RPMI. Solutions were prepared by adding 2g of WF or 2% of Corid in 98mL of RPMI, 1mL DMSO, and 1mL of Gibco Anti-Anti. CT exposure occurred where a 12 well plate, each solution was tested in triplicate by adding 200µL of extract and ~7000 oocysts per well. Plates were incubated at 27ºC for 24 h. Well contents were removed, washed, and concentrated to 200 µL. Oocysts were counted using 20 µL of trypan blue mixture and a hemocytometer. A bulk fecal sample with 4110 eggs per gram(EPG) was divided into 20 replicates of 9g each to prepare larval cultures. Treatment groups (n=5) were 5g, 3g, 1g, and 0g of WF(cabernet franc) in each sample. The WF mixture was then mixed in 125ml cups with water and vermiculite. The cups were covered with cheesecloth, inverted, and placed into 250ml tri-corner cups containing ~70ml of water. Cultures were incubated at room temp for 2 weeks. Larvae were collected by flooding cultures with warm water and concentrated down to 100-200 µL over a 3 day period by allowing larvae to sediment and removing the supernatant. The final larval pellet was mixed, removed and placed on a microscope slide with Lugol’s iodine. All larvae found were counted. Wine flour had no effect on oocyst excystation. The number of sporulated oocysts with unexcysted sporozoites was expected to increase with increasing concentrations of WF; however, that did not occur. WF did have an impact on development and survival of GI nematode larvae in feces. The number of surviving larvae was expected to decrease with increasing amounts of wine four, which was observed in this study.

Not Submitted

Funder Acknowledgement(s): Thank you to the LSU Ronald E. McNair Scholar?s, LS-LAMP LSU program and LSU IMSD program.

Faculty Advisor: Dr. James E. Miller, jmille1@lsu.edu

Role: I went to the farm and collected fecal samples. I made 2%, 0.2%, 0.02%, and 0.002% solutions of Cab Franc, Cab Sauv, Amprolium(Corid), and a negative control of RPMI. I Conducted CT exposure which included adding 200µL of extract and ~7000 oocysts per well in triplicated 12 well plate and then counted oocyst using a hemocytometer. I prepared larval cultures by making a bulk fecal sample with 4110 eggs per gram(EPG) and diving it into 20 replicates. I counted larvae using a two-chamber McMaster slide.