In vitro Evaluation of the Nutraceutical Potential of Theobroma cacao pod Husk and Leaf Extracts for Small Ruminants.

PURPOSE: Some agroindustrial by-products could be used as nutraceutical materials for small ruminants helping with their nutrition while controlling their gastrointestinal nematodes. This study evaluated the potential in vitro nutraceutical value of pod husks and leaves of three varieties of Theobroma cacao using two Haemonchus contortus isolates with different polyphenol susceptibility. METHODS: Leaves and husks from three T. cacao varieties (AZT, CAL and CEY) were evaluated for their bromatological composition, in vitro dry matter digestibility and polyphenol content. Acetone:water extracts (70:30) of each plant variety were evaluated using the egg hatch and larval exsheathment inhibition tests, using two isolates (FESC and PARAISO) of H. contortus. Effective concentrations 50% (EC50) were determined for both tests. The role of polyphenols was confirmed using polyvinylpolypyrrolidone. L3 exposed to CAL leaf extract were submitted to transmission electron microscopy. RESULTS: Both plant materials showed a good nutritional value to complement protein-rich diets for small ruminants. Extracts inhibited exsheathment of H. contortus L3 more effectively than the egg hatching, and the leaf extracts were more active than husk extracts in the L3 exsheathment inhibition. The FESC isolate was more sensitive to extracts. Polyphenols blocked exsheathment inhibition of leaf extracts. Structural damage was observed in the sheath and muscles of L3 exposed to CAL leaf extracts. CONCLUSION: The two T. cacao materials tested showed their potential to be used as ruminant feeds. Extracts affected H. contortus by blocking L3 exsheathment, particularly with the leaf extracts. The in vivo nutraceutical value should be confirmed in small ruminants.

Isolation of pure Trichostrongylus colubriformis strains from naturally infected sheep using two methodologies.

Two methodologies were tested to isolate pure Trichostrongylus colubriformis strains from naturally infected sheep. Also, the in vitro susceptibility status to commercial anthelmintic (AH) drugs was determined in these strains. These methods were performed in a tropical region of Mexico where naturally infected sheep and goats host Haemonchus contortus, T. colubriformis and Oesophagostomum columbianum. For the first strain, a group of 6 naturally infected lambs from the "Paraiso" farm were treated with closantel (subcutaneous (SC), 10 mg/kg). On day 10 post-treatment, the eggs per gram (EPG) of faeces were determined with the McMaster technique. The faeces from the two lambs with the highest EPG were used for coprocultures (4 days, 28 °C). The L3 larvae were recovered and identified as T. colubriformis (69%) and O. columbianum (31%). The latter was removed by 10-day refrigeration (4-5 °C) followed by sieving (25 µm), resulting in 100% T. colubriformis (PARAISO strain). The second strain was isolated using repeated doses of levamisole (LEV, SC 7.5 mg/kg) in an 8-year-old sheep. The sheep had 1700 EPG before the LEV treatments and maintained 1300 EPG after both LEV treatments (day 10). The coproculture (4 days, 28 °C) after the second treatment contained 100% T. colubriformis (FMVZ-UADY strain). The in vitro AH susceptibility was determined using the egg hatch test for benzimidazole (BZ), and the larval migration inhibition test for ivermectin (IVM) and LEV. The PARAISO strain was BZ- and LEV-susceptible, and IVM-resistant. Meanwhile, the FMVZ-UADY strain was BZ- and IVM-susceptible, and LEV-resistant. The conditions where these two protocols could be used in other parts of the world were discussed.

Optimal age of Trichostrongylus colubriformis larvae (L3) for the in vitro larval exsheathment inhibition test under tropical conditions.

This study identified the optimal age of Trichostrongylus colubriformis larvae (L3) under tropical conditions for the in vitro evaluation of plant extracts using the larval exsheathment inhibition test (LEIT). Two T. colubriformis isolates with different anthelmintic (AH) susceptibility status were used for this study. The L3 of both isolates were maintained on refrigeration (4-5 °C) until use. For the LEIT, the isolates were tested every week during 16 weeks, using a stock solution of Acacia pennatula acetone:water extract at different dilutions (80-1200 µg/mL). Respective positive controls (levamisole 12.5 mM) and negative controls (PBS) were included. Effective concentrations 50 % (EC50), 90 % (EC90) and respective 95 % confidence intervals (95 %CI) for exsheathment inhibition were calculated at 60 min after exposure to chlorine solution. Motility of L3 (migration percentages (M%)) was recorded weekly using the larval migration test (LMT) as an indicator of L3 fitness over time. No correlation was found between L3 age or M% and the extract's EC50 or EC90 values. However, the EC50 values for the A. pennatula extract ranged from 80 to 200 µg/mL from weeks 2-10. Beyond week 12, larval exsheathment was irregular, with higher EC50 and EC90 values and wider 95 %CI. The M% decreased below 85 % on week 7 for Paraiso isolate, and on week 10 for FMVZ isolate. A linear negative relationship was observed between the age of L3 and M% for both T. colubriformis isolates. The relationship (slope) for both isolates was similar therefore a single linear equation was estimated describing all M% data (r2 = 0.771, df = 164, P < 0.05). Thus, when using these T. colubriformis isolates under our tropical conditions for the evaluation of AH activity of plant extracts with LEIT, the optimal age of L3 is between weeks 2-7, when M% remained above 85 %. The latter may ensure consistent and reproducible exsheathment results for T. colubriformis. Each laboratory must identify optimal conditions to perform the LEIT.