Effect of using redberry juniper (Juniperus pinchotii) to reduce Haemonchus contortus in vitro motility and increase ivermectin efficacy.

A modified larval migration inhibition assay was used to determine if redberry juniper (Juniperus pinchotii Sudw.) can reduce Haemonchus contortus in vitro motility and increase ivermectin (IVM) efficacy. Ruminal fluid was mixed with buffer solution and either no material (CNTL) or Tifton 85 Bermudagrass hay (T85), dried juniper (DRY), fresh juniper (FRE), or distilled juniper terpenoid oil (OIL) to make treatment solutions and anaerobically incubated for 16 h. For Trial 1, larvae were incubated in CNTL, T85, DRY, or IVM. During Trial 2, larvae were incubated in CNTL, DRY, FRE, or OIL for 4h. Trials 3 (CNTL or OIL) and 4 (CNTL, DRY or FRE) evaluated larvae after incubation in treatment solution for 2h, then incubated an additional 2h in various IVM doses (0, 0.1, 1, 3, and 6 µg/mL IVM) and placed onto a screen. Larvae that passed through the 20-µm screen within a 96-well plate were considered motile. Larvae incubated in CNTL or T85 had similar (P=0.12) motility, but larvae incubated in DRY were less (P<0.02) motile than larvae incubated in CNTL or T85 (Trial 1). During Trial 2, adding DRY, FRE, or OIL reduced (P<0.001) larval motility as compared to CNTL. A treatment×IVM dose interaction (P=0.02) was observed during Trial 3, due to OIL unexpectedly decreasing IMV efficacy at IVM concentrations of 1 (P=0.07), 3, and 6 (P<0.002)µg/mL. No treatment×IVM dose interaction (P=0.57) was observed during Trial 4, but larvae incubated in DRY had less (P<0.004) total motility than larvae incubated in CNTL or FRE. Juniper forage material reduced in vitro H. contortus larval motility, but IVM efficacy was increased only by initially incubating larvae in DRY.

A modified in vitro larvae migration inhibition assay using rumen fluid to evaluate Haemonchus contortus viability.

Anthelmintic effects of plant secondary compounds may be occurring in the rumen, but in vitro larvae migration inhibition (LMI) methods using rumen fluid and forage material have not been widely used. Forage material added to an in vitro system can affect rumen pH, ammonia N, and volatile fatty acids, which may affect larvae viability (LV). Validating a LMI assay using rumen fluid and a known anthelmintic drug (Ivermectin) and a known anthelmintic plant extract (Quebracho tannins; QT) is important. Rumen fluid was collected and pooled from 3 goats, mixed with buffer solution and a treatment (1 jar/treatment), and placed into an anaerobic incubator for 16h. Ensheathed larvae (<3 months old) were then anaerobically incubated with treatment rumen fluid for 2, 4, or 16h depending on the trial. Larvae (n=15-45) were then transferred onto a screen (n=4-6 wells/treatment) within a multi-screen 96-well plate that contained treatment rumen fluid. Larvae were incubated overnight and those that passed through the 20-µm screen were considered viable. Adding dry or fresh juniper material reduced (P<0.05) pH, ammonia N, and isobutyric, butyric, isovaleric, and valeric acids, and increased (P<0.001) acetic, propionic, and total VFA. Including 4.5% (w/v) polyethylene glycol (PEG) in rumen fluid mixture with or without forage material reduced (P<0.01) LV. However, LV was similar at all PEG concentrations tested (0-2%, w/v; 89.4, 78.9, 76.5, 75.5, and 77.5% viable). Q. tannin concentrations from 0 to 1.2% (w/v) quadratically reduced (P<0.001) LV; 89.4, 65.5, 22.8, and 9.2%. Ivermectin concentrations from 0 to 15µg/mL quadratically reduced (P<0.001) LV; 90.2, 82.6, 73.6, 66.3, 51.9, 56.5, 43.5, 41.9, 29.3, and 19.9% viable, respectively. Effects of altering in vitro rumen fluid pH, ammonia N, and VFA and using PEG when evaluating LV need to be further investigated. In vitro rumen fluid assays using QT and Ivermectin resulted in decreased LV, validating the efficacy of this technique for measuring Haemonchus contortus larval viability.