Data inconsistencies have been reported with the larval feeding inhibition assay (LFIA), an in vitro parasitological technique which measures the reduction of food ingestion by first stage larvae (L1) incubated in serial dilutions of a test substance. Possible factors that can account for this observed variation in results include: parasite (species/strain); material tested; or season. There is thus need to optimise LFIA to permit intra and inter-laboratory comparison of results. We investigate here, if changes in EC50 values occur over the patency phase of a nematode species using two test substances with LFIA conducted in winter. Weekly rectal faecal samples collected during the patency period from a 4-month-old Jersey calf previously orally infected with L3 larvae of Cooperia onchophora, were used for the LFIA with subsamples used for faecal egg counts (FEC) by McMaster Technique. Serial dilutions (range: 0-160 μg/ml) in water of lyophilised sainfoin Cotswold Common Var; 12.5 g/100g of freeze-dried acetone water extract and epigallocatechin gallate (EGCG, range: 0-320 μg/ml), were used to incubate 100 L1/100 μl in Eppendorf tubes. Also, controls consisting of L1 in purified water (negative controls) or L1 in aqueous ivermectin (40 μg/ml; positive controls) were included. Each test concentration/control was prepared in triplicates. The EC50 values were determined weekly over four weeks starting two weeks after inoculation. Eggs started to appear in the faeces of calf 3 wks post infection and FEC varied considerably, peaking (3035 eggs per gram; epg) at end of the third week and decreased thereafter to almost zero by the end of the seventh week. Dose dependent responses in feeding behaviour of larvae were generally observed in higher concentrations but also inconsistent results reflecting the erratic larval feeding in some concentrations. Negative controls varied (43-83 %) in their feeding activity whereas positive controls mostly resulted in almost complete (97-100 %) feeding inhibition. The EC50 values obtained for incubating larvae in sainfoin extract over the 4 week period ranged from 12.2 to 58.9 μg/ml, whereas those obtained for EGCG showed a higher variation (39.2-176.8 μg/ml). No obvious patterns to changes in EC50 values indicated a systematic effect of time after infection on LFIA results. This study also highlighted the considerable day-to-day variation possible in LFIA results.
7th Novel Approaches To the Control of Helminths of Livestock: Bridges Between Scientific Advances and Farm Development, 2013
Optimisation; In vitro; Larval feeding inhibition assay; Variation
Main Research Area:
7th Novel Approaches to the Control of Helminths of Livestock (CAPARA 2013)