The in vitro rumen exsheathment test for studying the effect of plant extracts on the exsheathment of Haemonchus contortus infective larvae.

This study applied the in vitro rumen exsheathment test (IVRET) to evaluate the exsheathment kinetics of Haemonchus contortus infective larvae (L3) incubated in ruminal liquor (RL) containing acetone:water extracts of Acacia pennatula (AP), Gymnopodium floribundum (GF), Havardia albicans (HA) or Lysiloma latisiliquum (LL). The role of polyphenols in the biological activity of the evaluated extracts was also determined. Larvae were incubated in RL either alone or added with a different plant extract (AP, GF, HA, or LL) at 1200 µg/mL. Polyethylene glycol (PEG) was added to block polyphenols in each treatment (RL+PEG, AP+PEG, GF+PEG, HA+PEG, and LL+PEG). After incubation times of 0, 1, 3, 6, 9, and 24 h, the exsheathment process was stopped to count the number of ensheathed and exsheathed L3. A Log-Logistic model was used to determine the L3 exsheathment kinetics in the different RL treatments. The inflection point of the respective kinetic curves, which indicates the time to reach 50 % exsheathed L3 (T50), was the only parameter that differed when comparing the exsheathment models (99 % probability of difference). The T50 values obtained for GF, HA, and LL treatments (T50 = 7.11 - 7.58 h) were higher in comparison to the T50 of RL (5.72 h) (≥ 70 % probability of difference). The L3 incubated in RL added with GF, HA, and LL extracts delayed their exsheathment at 3 and 6 h of incubation (28.71 - 48.06 % exsheathment reduction) compared to the RL treatment. The T50 value for AP, AP+PEG, GF+PEG, HA+PEG, and LL+PEG were similar to RL and RL+PEG (T50 = 5.34 - 6.97 h). In conclusion, the IVRET can be used to identify plants with the potential to delay the exsheathment of H. contortus L3 in the ruminal liquor. The acetone:water extracts of G. floribundum, H. albicans, and L. latisiliquum delayed the T50 of H. contortus exsheathment, which was evident at 3 and 6 h of incubation in ruminal liquor. The observed exsheathment delay was attributed to the polyphenol content of the extracts.

Adapting the in vitro rumen incubation method to evaluate the effect of a plant extract on the exsheathment inhibition of Haemonchus contortus infective larvae.

This study adapted the in vitro rumen incubation (IVRI) method to evaluate the biological activity of a Gymnopodium floribundum leaves extract against the exsheathment of Haemonchus contortus infective larvae (L3), and to determine the role of plant polyphenols on the biological activity. The incubation protocol followed the IVRI method, adding polyethylene glycol (PEG) as a polyphenol-blocking agent. The L3 were incubated in ruminal liquor (RL), ruminal liquor with PEG (RL+PEG), ruminal liquor with G. floribundum extract (RLE), and ruminal liquor with G. floribundum extract and PEG (RLE+PEG). Incubation condition controls included phosphate buffered saline (PBS), PBS with PEG (PBS+PEG), incubation medium (without ruminal liquor) (IM), and incubation medium with PEG (IM+PEG). The L3 were recovered after incubation times of 0, 1, 3, 6, 9, and 24 h (39 °C). The respective L3 exsheathment kinetics were estimated for the different treatments (RL, RL+PEG, RLE, and RLE+PEG) using Log-Logistic models. The parameters of the different models were compared to determine the impact of the extract, with or without PEG, on the L3 exsheathment kinetics. The exsheathment in PBS and PBS+PEG remained < 2.71% at each incubation time. The exsheathment in IM and IM+PEG reached 13.58% and 17.18% at 24 h, respectively. The exsheathment percentages for RLE were lower than those for RL at 3, 6 and 9 h of incubation. The inflection point, indicating the time required to reach 50% of the maximal exsheathment (T50), was the only parameter that differed between the ruminal liquor models. The T50 in RLE (7.106 h) was higher than the values obtained for RL (5.385 h) and RL+PEG (4.923 h) (99.99% probability of being different). Such delay resulted in a reduction of exsheathment in RLE of 62% at 3 h, 38% at 6 h, and 12% at 9 h, relative to RL values. When PEG was added with the extract (RLE+PEG), the T50 (5.045 h) was similar to that of RL and RL+PEG. The IVRI method was adapted as an in vitro rumen exsheathment test (IVRET). The IVRET showed that H. contortus L3 exposed to G. floribundum extract delayed their exsheathment kinetics at different time points. The exsheathment delay was attributed to the polyphenol content of the extract.

Two in vitro anthelmintic assays of four Paraguayan medicinal plants for proof of concept of the role of polyphenols in their biological activities and LC-HRMS analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Gastrointestinal nematodes (GIN) control in small ruminants has relied on the systematic use of synthetic anthelmintics (AH), their effectiveness has been progressively decreasing due to the rise and diffusion of anthelmintic resistances. The most prevalent genera affecting small ruminants were Haemonchus spp., and Trichostrongylus spp. The investigation of new anthelmintics in plants is a highly studied option, especially when it is linked to ethnobotanical knowledge and phenolic compounds. THE AIMS OF OUR STUDY: Four medicinal plants mentioned in traditional medicine were selected to evaluate their anthelmintic proprieties at different stages of the life cycle of GIN, namely: Kyllinga odorata Valh., Cassia occidentalis L., Artemisia absinthium L, and Verbena litoralis Kunth and to explore the role of polyphenols in the AH activity. MATERIALS AND METHODS: To evaluate the anthelmintic activity in this study, two models of GIN species, namely Haemonchus contortus (Hc) and Trichostrongylus colubriformis (Tc) were selected and tested on two in vitro assays: 1) Larval Exsheathment Inhibition Assay (LEIA) and, 2) Egg Hatch Assay (EHA). To explore the role of tannins and polyphenols in AH activity by comparing the effects of LEIA and EHA with or without polyvinylpolypyrrolidone (PVPP) and to characterize the phytochemical composition of the most active plants using ultra-high performance liquid chromatography (UHPLC) coupled with high-resolution mass spectrometry (HRMS). RESULTS: C. occidentalis exhibited the highest activity on LEIA (EC50 = 250.42-41.80 µg/mL) and A. absinthium on egg hatching processes (EC50 = 121.70-137.34 µg/mL) in both species of GIN. The inhibition in the development of eggs was from 67.70% to 96.36% on H. contortus, and from 78.87% to 99.65% on T. colubriformis. At the maximal dose, Additionally, it was observed that the AH on eggs varies according to the GIN species: on H. contortus the extracts tested blocked the formation of larvae Ovicidal Effect (% higher OE) and on T. colubriformis they blocked the appearance of L1 larvae, Larvae Failing Eclosion (% higher LFE). After PVPP, a reduction in AH activity on LEIA and EHA was noted, especially with C. occidentalis (87.20-67.00% of larvae exsheathment, (p < 0.05) and 40.51-24.96% of egg hatching, (p > 0.05) of both parasite species. Nine putative features were identified using HRMS and MS/MS after addition of PVPP. CONCLUSIONS: The present study demonstrated that C. occidentalis, A. absinthium, and K. odorata, which parts have been traditionally used as medicinal plants are a valuable source of active compounds with anthelmintic activity. The medicinal use of these plants against GIN parasites was proven by in vitro analysis. Therefore exploration of the secondary metabolites of these plant extracts and testing of isolated fractions of active compounds under in vivo experiments are planned and represent a specific challenge for alternative drug research. Regarding the PVPP, in this study we hypotheses about the standard doses it was not able to completely absorb the polyphenols of extracts of K. odorata, C. occidentalis, and A. absinthium, which would lead to more studies to evaluate the role of this product in the absorption of phenolic compounds.

Scanning electron microscopy of different vulval structures in a Mexican Haemonchus contortus isolate.

Haemonchus contortus is a parasite species that affects the health and production of grazing small ruminants in different parts of the world. Scanning electron microscopy (SEM) is an important tool for the study of parasites' morphology and taxonomy as it generates images that appear 3D and are generally easier to interpret than optical microscopy images. This study used the SEM to describe the vulval types of adult H. contortus from a Mexican isolate. A total of 14 adult H. contortus females were obtained from two artificially infected goats. Females were fixed and processed by critical point drying and observed with SEM. A collection of SEM images was obtained from these parasites and those images were used to identify the structures previously described by optical microscopy studies. Two different types of vulval structures were described in this Mexican H. contortus isolate: Type 1 (vulval flap), Type 2 (epiptygma). An unusual vulval structure was reported in a single individual. The Type 1 included vulval flaps of different sizes and spatial dispositions, as well as one or more knobs in different positions around the vulva. The Type 2 shows differences in the epiptygma. The present study suggests that the Mexican H. contortus isolate used in donor animals possess intraspecific polymorphism in vulval structures.

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.

Tannin supplementation modulates the composition and function of ruminal microbiome in lambs infected with gastrointestinal nematodes.

This study was carried out to evaluate the effects of tannin supplementation on ruminal microbiota of sixteen lambs infected and non-infected with Haemonchus contortus and Trichostrongylus colubriformis. Animals were fed with hay, concentrate and supplemented with Acacia mearnsii (A. mearnsii). The animals were divided into four treatments: two control groups without infection, either receiving A. mearnsii (C+) or not (C-), and two infected groups, one with A. mearnsii (I+) and another without A. mearnsii (I-). Ruminal short-chain fatty acids (SCFA) and metagenome sequencing of ruminal microbiota were used to evaluate the effect of tannin and infection on ruminal microbiome. For SCFA, differences were observed only with A. mearnsii. Total SCFA and acetate molar percentage were decreased in C+ and I+ (P<0.05). Butyrate, valerate and isovalerate were higher in lambs that received A. mearnsii in the diet (P<0.05). The infection changed the microbiome structure and decreased the abundance of butyrate-producing microorganisms. In addition, A. mearnsii supplementation also affected the structure the microbial community, increasing the diversity and abundance of the butyrate-producing and probiotics bacteria, amino acid metabolic pathways, purine, pyrimidine and sphingolipid metabolism. Together, our findings indicate that A. mearnsii supplementation modulates important groups related to nitrogen, amino acid, purine and pyrimidine metabolism, in rumen microbiome, affected by gastrointestinal nematodes infection in lambs.

Evaluation of targeted drenching using Famacha method in Creole goat: reduction of anthelmintic use, and effects on kid production and pasture contamination.

Haemonchus infection was monitored for 2 years (six kidding periods) in a Creole goat flock grazing under oceanic-tropical climate. Two hundred and thirty individual does were involved from late pregnancy to weaning. Anaemia diagnoses using Famacha method and packed cell volume (PCV) were compared. The best agreement (Kappa=0.33) was found if anaemia was declared when PCV values fell to 16 or below and Famacha score was 4 or 5. Drenching policy according to Famacha method was compared to systematic drenching policy. Using the Famacha method allowed a dramatic decrease in anthelmintic use during the periparturient period (0.57 individual dose instead of three doses for the control). The proportion of the nematode population on the pasture not derived from previously-treated goats (in refugia) was estimated to about 79% (65-90%) of the pasture contamination derived from the Famacha group. On the average, goats which needed to be drenched produced less than the control or undrenched goats (kid average daily gain was decreased by about 15%). The repeatability of the need for drenching individual does was estimated to 0.41. The older goats or the goats in poorer body condition at kidding needed more drenching than the younger animals and the animals in good body condition. Consequently the Famacha method may be used as an additional tool for the culling management.