In vitro screening of the anthelmintic efficacy of birdsfoot trefoil commercial varieties and cultivars against ovine Haemonchus contortus

Abstract Text: Purpose: Some forages containing condensed tannins (CT), also called proanthocyanidins (PAC), suppress gastrointestinal nematode (GIN) infections in small ruminants.  The objective of this study was to investigate the anthelmintic potential of 51 commercial varieties and cultivars of birdsfoot trefoil (BFT) against Haemonchus contortus.  The anti-parasitic activity of BFT proanthocyanidin extract (BFT-PAC) and BFT aqueous extract (BFT-AqE) was tested using the following in vitro assays: 1) egg hatching and viability of L1 H. contortus larvae and 2) exsheathment of L3 H. contortus larvae.  Methods:  BFT powder of each variety or cultivar was analyzed for CT content (mg/g) by the DMAC (4-(dimethylamino)cinnamaldehyde) method.  BFT-PAC was prepared by isolating PAC extract from the BFT powder using solid-phase chromatography.  BFT-AqE was prepared by soaking BFT powder in water at room temperature for 24 hours.  The plant matter was then removed, leaving an aqueous extract.  1) In vitro egg hatch and viability of L1 larvae: H. contortus eggs were isolated from fresh feces and exposed to varying concentrations of BFT extracts for 24 hours.  The percentage of hatched eggs and L1 larval mortality (based on motility) were determined.  2) In vitro exsheathment: 2,000 H. contortus L3 larvae were incubated in varying concentrations of BFT extracts for 24 hours, followed by exsheathment using CO₂.  The percentage of exsheathed larvae (based on absence of sheath) was determined.  Results:  Condensed tannin content ranged between 1.5 and 63.8mg/g across 51 varieties and cultivars.  Inhibition of egg hatch and larval mortality was observed with incubation in BFT-AqE, however the concentration at which this inhibition and mortality was most effective varied among varieties and cultivars: At 3mg/mL, percent inhibition of egg hatch and L1 mortality spanned between 0%-100% across 51 varieties and cultivars tested.  Results for incubation in BFT-PAC and results for exsheathment are pending.  Conclusion:  Preliminary results indicate that commercial varieties and cultivars of BFT-AqE inhibited egg hatch and increased larval mortality, but the degree of inhibition and mortality varied.  Additional results testing BFT-PAC and testing exsheathment will provide further information about the anthelmintic efficacy of commercial varieties and cultivars of BFT for small ruminant GIN control.

Keywords: small ruminant, sheep, condensed tannin