Curcumin-PVP improves the in vitro efficacy of ivermectin against resistant and susceptible Haemonchus contortus.

Ivermectin (IVM) resistance in parasitic nematodes such as Haemonchus contortus has spurred a search for substances that help to recover its efficacy. One potential agent is the natural product curcumin (CUR). In this study, CUR was combined with polyvinylpyrrolidone (PVP) (CUR/PVP) to improve its solubility and biological applicability. This study determined the effect of CUR preincubation on the effective concentration 50% (EC50) of IVM in three H. contortus isolates with different susceptibilities to IVM. The IVM EC50 was determined for three H. contortus isolates with different IVM susceptibilities using the larval migration inhibition (LMI) test. The three isolates were (i) PARAISO (IVM resistant), (ii) FMVZ-UADY (IVM susceptible), and (iii) CENID-SAI INIFAP (reference IVM susceptible). The L3 of each isolate were preincubated for 3 h with one of three concentrations of CUR (µg curcumin/mL): CONC-1 (3.67), CONC-2 (5.67), or CONC-3 (8.48). Corresponding controls were performed without CUR. The EC50 of IVM was determined for each isolate after they were exposed to the different CUR concentrations. The EC50 of IVM differed between the isolates PARAISO > FMVZ-UADY > CENID-SAI INIFAP (P < 0.05). The CUR preincubation at CONC-1 did not decrease the EC50 of IVM for any of the three isolates, suggesting a hormetic effect. By contrast, CUR preincubation at CONC-2 or CONC-3 decreased the IVM EC50 for the PARAISO isolate (P < 0.05) compared with the reference isolate and reduced the EC50 of IVM for the FMVZ-UADY and CENID-SAI INIFAP isolates below the EC50 for the CENID-SAI INIFAP isolate without CUR preincubation. In conclusion, preincubation of H. contortus L3 with CUR reduced the EC50 of IVM for field isolates classified as resistant and susceptible to IVM. The CUR preincubation reduced the IVM resistance factor in the different isolates tested.

Cytotoxic Furanoditerpenes from the Sponge Spongia tubulifera Collected in the Mexican Caribbean.

Two new spongian furanoditerpenes, 3ß-hydroxyspongia-13(16),14-dien-2-one (1) and 19-dehydroxy-spongian diterpene 17 (2), along with five known terpenes, the spongian furanoditerpenes 9-nor-3-hydroxyspongia-3,13(16),14-trien-2-one (3), 3ß,19 dihydroxyspongia-13(16),14-dien-2-one (epispongiadiol) (4) and spongian diterpene 17 (5), the furanoditerpene ambliol C (6), and the sesterterpene scalarin (7), were isolated from the methanolic extract of the sponge Spongia tubulifera, collected in the Mexican Caribbean. The planar structures of the new compounds were elucidated by 1D/2D NMR and IR spectroscopic analysis, high resolution electrospray mass spectrometry (HRESIMS), and comparison of their spectral data with those reported in the literature. Absolute configurations were determined by comparison of the experimental electronic circular dichroism (ECD) spectrum with those calculated by time-dependent density functional theory (TDDFT). Compounds 1, 4, and 6 displayed weak cytotoxic activity against different human tumour cell lines.

Salivary tannin-binding proteins are a pervasive strategy used by the folivorous/frugivorous black howler monkey.

Dietary tannins can affect protein digestion and absorption, be toxic, and influence food selection by being astringent and bitter tasting. Animals that usually ingest tannins may regularly secrete tannin-binding salivary proteins (TBSPs) to counteract the negative effects of tannins or TBSPs production can be induced by a tannin-rich diet. In the wild, many primates regularly eat a diet that contains tannin-rich leaves and unripe fruit and it has been speculated that they have the physiological ability to cope with dietary tannins; however, details of their strategy remains unclear. Our research details the salivary protein composition of wild and zoo-living black howler monkeys (Alouatta pigra) feeding on natural versus manufactured low-tannin diets, and examines differences in TBSPs, mainly proline-rich proteins (PRPs), to determine whether production of these proteins is dependent on the tannin content of their food. We measured the pH, flow rate, and concentration of total protein and trichloroacetic acid soluble proteins (an index of PRPs) in saliva. Howler monkeys produced slightly alkaline saliva that may aid in the binding interaction between tannin and salivary proteins. We used gel electrophoresis to describe the salivary protein profile and this analysis along with a tannin-binding assay allowed us to detect several TBSPs in all individuals. We found no differences in the characteristics of saliva between wild and zoo-living monkeys. Our results suggest that black howler monkeys always secrete TBSPs even when fed on foods low in tannins. This strategy of constantly using this salivary anti-tannin defense enables them to obtain nutrients from plants that sometimes contain high levels of tannins and may help immediately to overcome the astringent sensation of their food allowing howler monkeys to eat tanniferous plants.

Rumen function in vivo and in vitro in sheep fed Leucaena leucocephala.

The effect of Leucaena leucocephala inclusion in sheep diets upon rumen function was evaluated. Nine Pelibuey sheep, 32.6 ± 5.33 kg live weight (LW), fitted with rumen cannula were used. A complete randomized block design was employed. Two experimental periods of 60 days each, with 60-day intervals between them, were used. Experimental treatments were as follows (n = 6): T1 (control), 100 % Pennisetum purpureum grass; T2, 20 % L. leucocephala + 80 % P. purpureum; T3, 40 % L. leucocephala + 60 % P. purpureum. In situ rumen neutral detergent fiber (aNDF) and crude protein (CP) degradation, dry matter intake (DMI), volatile fatty acids (VFA) production, estimated methane (CH4) yield, rumen pH, ammonia nitrogen (N-NH3), and protozoa counts were measured. The aNDF in situ rumen degradation of P. purpureum and leucaena was higher (P < 0.05) in T2 and T3. Leucaena CP degradation was higher in T2 and T3 but for P. purpureum it was only significantly higher in T3. Leucaena aNDF and CP degradation rate (c) was 50 % higher (P < 0.05) in T2 and T3, but only higher in T3 for P. purpureum. Voluntary intake and rumen (N-NH3) was higher in T2 and T3 (P = 0.0001, P = 0.005, respectively). Molar VFA proportions were similar for all treatments (P > 0.05). Protozoa counts and in vitro gas production (48 h) were lower in T2 and T3 (P < 0.05, P < 0.0001). Estimated methane yield (mol CH4/day) was higher in sheep fed leucaena (P < 0.0001). However, CH4 yield relative to animal performance (mol CH4/g LW gain) was lower in T2 and T3 (P < 0.0001). In summary, these results indicate that including L. leucocephala in sheep diets did not modify rumen fermentation pattern (same VFA ratios) nor reduce the amount of CH4 per unit of DMI (mol CH4/g DMI). However, leucaena inclusion does increase rumen N-NH3, aNDF and CP digestibility, and voluntary intake.

Relevance of Individual Data When Assessing the Gastrointestinal Nematode Infection Level, Nutritional and Productive Variables in a Tropical Farm Context: The Median Isn't the Message.

We evaluated the relationship between individual and herd GIN infection level, nutrition, production performance and anemia parameters in a tropical farm context. Fifty-four female goats were monitored to assess their body condition score (BCS, nutritional status indicator), live weight (LW) and LW gain (LWG, both used as production level indicators), FAMACHA© and hematocrit (HT, both used as anemia indicators). Goats browsed for 4 h in a tropical forest and received balanced feed and chopped grass. The eggs per gram of feces (EPG) indicated the GIN burden, with fecal samples obtained at 7:00 (AM) and 15:00 h (PM.) from each goat at six sampling points during the study. The variables and their relationship with GIN burdens were analyzed using Kruskall-Wallis, ANOVA and Friedman tests and Spearman correlations. The fecal samples obtained in the AM and PM can be equally representative of parasitic burdens (similar and highly correlated). However, the EPG of individual goats from periods of 30 days apart can be considered independent. The BCS and LWG varied between sampling times (p < 0.05), whereas EPG, LW and HT did not (p > 0.05). The GIN burden was negatively correlated with HT and BCS (-0.21, p = 0.01 for each one). The individual pattern of infection demonstrates the true impact of GINs on their hosts. Additionally, feeding and nutritional status may present important variations influencing the performance of the goats more than the impact of GINs under the farm conditions of the present study. However, GIN infection contributed to the variation in goat health and productivity in this tropical farm.

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.

Comparison of P-glycoprotein gene expression of two Haemonchus contortus isolates from Yucatan, Mexico, with resistant or susceptible phenotype to ivermectin in relation to a susceptible reference strain.

The variability in the expression of different P-glycoprotein (P-gp) genes in parasitic nematodes of ruminants such as Haemonchus contortus (Hco-pgp) may be caused by different factors including nematode biology, geographical region and anthelmintic pressure. This study analysed the relative expression level of 10 P-gp genes in two H. contortus (Hco-pgp) field isolates from Yucatan, Mexico: 1) PARAISO (IVM-resistant) and 2) FMVZ-UADY (IVM-susceptible). These isolates were compared with a susceptible reference isolate from Puebla, Mexico, namely "CENID-SAI". In all cases H. contortus adult males were used. The Hco-pgp genes (1, 2, 3, 4, 9, 10, 11, 12, 14 and 16) were analysed for each isolate using the RT-qPCR technique. The Hco-pgp expressions were pairwise compared using the 2-ΔΔCt method and a t-test. The PARAISO isolate showed upregulation compared to the CENID-SAI isolate for Hco-pgp 1, 3, 9, 10 and 16 (P < 0.05), and the PARAISO isolate showed upregulation vs. FMVZ-UADY isolate for Hco-pgp 2 and 9 (P < 0.05), displaying 6.58- and 5.93-fold differences (P < 0.05), respectively. In contrast, similar Hco-pgp gene expression levels were recorded for FMVZ-UADY and CENID-SAI isolates except for Hco-pgp1 (P <0.1), which presented a significant upregulation (6.08-fold). The relative expression of Hco-pgp allowed confirming the IVM-resistant status of the PARAISO isolate and the IVM-susceptible status of the FMVZ-UADY isolate when compared to the CENID-SAI reference isolate. Therefore, understanding the association between the Hco-pgp genes expression of H. contortus and its IVM resistance status could help identifying the genes that could be used as molecular markers in the diagnosis of IVM resistance. However, it is important to consider the geographic origin of the nematode isolate and the deworming history at the farm of origin.

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.

Nutrient Digestibility and Metabolizable Energy Content of Mucuna pruriens Whole Pods Fed to Growing Pelibuey Lambs.

The nutrient digestibility, nitrogen balance and in vivo metabolizable energy supply of Mucuna pruriens whole pods fed to growing Pelibuey lambs was investigated. Eight Pelibuey sheep housed in metabolic crates were fed increasing levels of Mucuna pruriens pods: 0 (control), 100 (Mucuna100), 200 (Mucuna200) and 300 (Mucuna300) g/kg dry matter. A quadratic (p<0.002) effect was observed for dry matter (DM), neutral detergent fibre (aNDF), nitrogen (N) and gross energy (GE) intakes with higher intakes in the Mucuna100 and Mucuna200 treatments. Increasing M. pruriens in the diets had no effect (p>0.05) on DM and GE apparent digestibility (p<0.05). A linear reduction in N digestibility and N retention was observed with increasing mucuna pod level. This effect was accompanied by a quadratic effect (p<0.05) on fecal-N and N-balance which were higher in the Mucuna100 and Mucuna200 treatments. Urine-N excretion, GE retention and dietary estimated nutrient supply (metabolizable protein and metabolizable energy) were not affected (p>0.05). DM, N and GE apparent digestibility coefficient of M. pruriens whole pods obtained through multiple regression equations were 0.692, 0.457, 0.654 respectively. In vivo DE and ME content of mucuna whole pod were estimated in 11.0 and 9.7 MJ/kg DM. It was concluded that whole pods from M. pruriens did not affect nutrient utilization when included in an mixed diet up to 200 g/kg DM. This is the first in vivo estimation of mucuna whole pod ME value for ruminants.

Evaluation of the metabolizable energy value for growing lambs of the Mucuna pruriens seed and the whole pod.

Whole pod and seeds of velvet bean (Mucuna pruriens) were included in diets for growing sheep used to validate previously estimated ME values of 9.7 MJ and 12.6 MJ for whole pod and seed respectively. Twenty-four lambs, 15 females and nine males of 18.7 ± 2.4 kg average weight, were allocated in three treatments using a completely randomized block design with eight replicates per treatment. Each group was given a diet with a ratio of 60% of Napier grass (Pennisetum purpureum) and 40% of a supplement with the addition of 0%, 50% whole pod or 66% of M. pruriens seeds, for TI, TII and TIII respectively. Diets were formulated to meet the requirements for 100-g daily live weight gain (LWG). The total dry matter intake (836 g a(-1) d(-1), forage + supplement), LWG (90 g a(-1) d(-1)) and feed conversion (9.66 kg DM/kg LWG) of lambs from TIII were lower (P < 0.05) compared to 941 g, 121 g, and 7.78 kg DM/kg LWG from TII and 976 g, 132 g and 7.50 kg DM/kg LWG from TI respectively. No difference was found (P > 0.05) between TI and TII in the three evaluated variables. The ME values of whole pod and seeds of M. pruriens used in this work were validated. It was concluded that M. pruriens can be included as a component in diets for growing sheep, as a partial replacement of conventional feedstuffs.

Selection of Forage Resources by Juvenile Goats in a Cafeteria Trial: Effect of Browsing Experience, Nutrient and Secondary Compound Content.

We evaluated the effect of browsing experience, nutritional quality and secondary compounds of forage resources, and the interaction between these factors on the selection and intake of goats in a cafeteria trial. Twelve juvenile Criollo goats from 7 to 9 months of age, weighing 22 ± 3 kg, were divided into two groups: (a) browser goats group (n = 6, BG), and (b) naïve goats group (n = 6, NG), formed according to their previous browsing experience (with and without, respectively). Animals were housed in individual pens. The cafeteria experiment lasted 21 days considering pen adaptation, foliage adaptation, and measurements, which included the selection index (SI) of experimental forage resources (Chesson's alpha) and their dry matter intake (DMI/Kg0.75), using a multiple Latin square design. Furthermore, correlation and regression analyses were used to assess the relationship between the aforementioned factors. The NG did not show any selection pattern, while the BG selected Piscidia piscipula and Senegalia gaumeri (p = 0.0002). The BG consumed smaller amounts of secondary compounds compared to NG (p = 0.0001). In the BG, the flavonoids affected negatively their selection (R2 = 97.51, p = 0.0001), while the DMI was affected by in vitro DM digestibility and flavonoids (R2 = 99.85; p = 0.0001). For the NG, the crude protein and organic matter contents were associated with DMI, but none had a significant relationship with SI. The BG selected and consumed forages with suitable nutritional quality avoiding those with high content of secondary compounds such as flavonoids. Conversely, NG did not show a clear pattern for their selection or intake.

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.

Small Ruminant Production Based on Rangelands to Optimize Animal Nutrition and Health: Building an Interdisciplinary Approach to Evaluate Nutraceutical Plants.

The plant kingdom can influence the productivity and health of herbivores at different levels. However, demonstrating this process in a scientific manner entails substantial endeavors from different disciplines. In the present review, we will describe the features of a native vegetation system traditionally used by small ruminants and use its particularities to build an interdisciplinary approach to evaluate the nutraceutical properties of plants. Initially, we will establish the context of the low deciduous forest (LDF), considering some botanical and nutritional aspects, as well as the presence of plant secondary compounds (PSC) and gastrointestinal nematodes (GIN). Furthermore, we will focus on coevolutionary aspects that undoubtedly shaped the plants-nutrients-PSC-GIN-herbivore relationship. In addition, the concept of nutraceutical will be discussed to provide clarity and aspects to be considered for their evaluation. Then, ethological, agronomical, nutritional, PSC, parasitological and animal species issues are deepened placing emphasis on methodological approaches. Special focus is given to condensed tannins, as they are the fourth largest group of PSCs and the most studied in livestock sciences. Validation of the nutraceutical properties of plants from native vegetation systems should be seen as a process derived from many scientific disciplines that feed into each other in a cyclic manner.

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.

Ultrastructural study of adult Haemonchus contortus exposed to polyphenol-rich materials under in vivo conditions in goats.

This study assessed the ultrastructural changes caused in adult Haemonchus contortus obtained from goats fed fodder based on polyphenol-rich plants Lysiloma latisiliquum or Onobrychis viciifolia or from goats drenched with quebracho extract, Schinopsis spp. The H. contortus were obtained from artificially infected goats used as models to investigate the anthelmintic effect of feeding or drenching with the polyphenol-rich materials. Nematode populations were exposed to polyphenol-rich plant materials by feeding host goats for 8 consecutive days (D28 to D35 post-infection) with (a) L. latisiliquum fodder at 800 g fresh basis/day, (b) O. viciifolia fodder offered ad libitum, and (c) drenched with a solution containing quebracho extract (90 g/day). Meanwhile, control H. contortus were obtained from goats fed polyphenol-free diets. The H. contortus specimens were recovered from the goats on D36 post-infection, and transmission electron microscopy (TEM) was used to identify ultrastructural changes. In vivo exposure to different polyphenol-rich plant materials caused vacuolization of the nematodes' intestinal, muscular and hypodermal cells. These alterations represent the first evidence of cell damage caused in H. contortus when hosts were fed or drenched with polyphenol-rich materials. Ultrastructural changes affecting several types of cells could explain modifications in worm motility and nutrition, eventually affecting H. contortus reproductive success. This study contributes to our understanding of the mechanisms of action of polyphenol-rich plants against H. contortus when given as nutraceuticals to goats.

TITLE: Étude ultrastructurale d'Haemonchus contortus adultes exposés in vivo à des matériaux riches en polyphénols chez les chèvres. ABSTRACT: Cette étude a examiné les lésions ultrastructurales provoquées chez les adultes d'Haemonchus contortus obtenus chez des chèvres alimentées par des fourrages riches en polyphénols, Lysiloma latisiliquum ou Onobrychis viciifolia, ou des chèvres recevant des extraits de quebracho, Schinopsis spp. Les H. contortus ont été obtenus à partir de chèvres artificiellement infestées pour vérifier les effets anthelminthiques de l'alimentation ou l'administration de ressources riches en polyphénols. Les populations de nématodes ont été exposées à des ressources végétales contenant des polyphénols chez des hôtes infestés recevant pendant 8 jours (J28 à J35 après infestation) : (a) un fourrage à base de L. latisiliquum (800 g/jour), (b) un fourrage à base de sainfoin (O. viciifolia) offert ad libitum, et (c) une solution d'extrait de quebracho (90 g/jour). Pendant ce temps, une population témoin d'H. contortus a été obtenue à partir de chèvres alimentées avec un régime à base de fourrages sans polyphénols. Des échantillons d'H. contortus ont été obtenus à partir des divers groupes de chèvres à J36 après infestation, et la microscopie électronique à transmission a été utilisée pour identifier les modifications ultrastructurales. L'exposition in vivo à différents matériaux végétaux riches en polyphénols chez l'hôte a provoqué chez les nématodes une vacuolisation des cellules intestinales, musculaires et hypodermiques. Ces altérations représentent la première preuve de lésions cellulaire provoquées chez H. contortus lorsque l'hôte reçoit des ressources riches en polyphénols. Les changements ultrastructuraux affectant divers types de cellules pourraient expliquer des modifications fonctionnelles de motilité et de nutrition des vers, et finalement modifier le succès reproducteur d'H. contortus. Cette étude contribue à la compréhension des mécanismes d'action des plantes contenant des polyphénols utilisées comme alicament contre H. contortus.

Evaluation of cinnamic acid and six analogues against eggs and larvae of Haemonchus contortus.

This study evaluated the in vitro anthelmintic (AH) activity of cinnamic acid and six analogues against eggs and larvae of Haemonchus contortus. Stock solutions of each compound (trans-cinnamic acid, p-coumaric acid, caffeic acid, trans-ferulic acid, trans-sinapic acid, 3,4-dimethoxycinnamic acid, and chlorogenic acid) were prepared in PBS:Tween-20 (1%) for use in the egg hatch test (EHT) and larval exsheathment inhibition test (LEIT) at different concentrations (25-400 µg/mL). The respective effective concentration 50% (EC50) values with 95% confidence intervals were estimated. Mixtures made of all cinnamic acid and its analogues as well as some selected individual compounds were also tested in the EHT. Only ferulic and chlorogenic acids showed AH activity in the EHT (EC50: 245.2 µg/mL (1.26 mM) and 520.8 µg/mL (1.47 mM), respectively) (P < 0.05). A higher EC50 (1628.10 µg/mL) of the mixture of cinnamic acid and its analogues was required to observe activity against eggs mostly blocking the larvae hatching. The analogues' mixtures tested were less active than ferulic or chlorogenic acid alone. The activity of ferulic and chlorogenic acids against eggs was associated with larvae failing to hatch, and the two compounds exhibited antagonistic effects when evaluated together. All standards had an EC50 lower than 0.42 mM in the LEIT. Caffeic acid had the best activity in the LEIT (EC50 0.04 mM), followed by ferulic acid (EC50 0.11 mM) (P < 0.05). There was no clear, definitive structure-activity relationship for these non-flavonoid polyphenols against eggs or larvae of H. contortus in vitro. This study is the first to directly evaluate cinnamic acid and its derivatives as active compounds against eggs and larvae of H. contortus.

Bio-guided fractionation to identify Senegalia gaumeri leaf extract compounds with anthelmintic activity against Haemonchus contortus eggs and larvae.

Small ruminants browsing in tropical forests readily consume the foliage of Senegalia gaumeri. A S. gaumeri methanol:water extract was recently shown to have ovicidal activity against Haemonchus contortus eggs in vitro. In the present study, the fraction of a S. gaumeri methanol:water extract with ovicidal activity against H. contortus eggs and the metabolites potentially involved in this activity were identified. Bio-guided fractionation of the S. gaumeri methanol:water extract identified high ovicidal activity (80.29%, EC50 = 58.9 µg/mL) in the non-polar sub-fraction P1. Gas chromatography-mass spectrometry (GC-MS) identified several fatty acids: pentacosane (18.05%), heneicosane (18.05%), triacontane (30.94%), octacosane (18.05%), and hexanedioic acid bis-(2-ethylhexyl) ester (32.72%). Purification of the polar components of sub-fraction P1 led to the identification of p-coumaric acid as a major constituent. In egg hatch tests, 400 µg/mL p-coumaric acid resulted in an ovicidal effect of 8.7%, a larvae failing eclosion effect of 2.9%, and of the emerged larvae (88.4%), many were damaged. In conclusion, the low AH activity of p-coumaric acid against H. contortus eggs indicates that it is not solely responsible for the ovicidal activity of sub-fraction P1 but might act in synergy with other compounds in this fraction. However, p-coumaric acid showed potential anthelmintic effects against the larval stage of H. contortus.

Assessing the efficacy of Duddingtonia flagrans chlamydospores per gram of faeces to control Haemonchus contortus larvae.

The aims were (a) to quantify the number of Duddingtonia flagrans chlamydospores per gram of faeces (CPG) recovered from sheep administered with different oral doses and, (b) to describe the relationship between CPG and eggs per gram of faeces (EPG) on the efficacy to reduce Haemonchus contortus infective larvae. Three doses of chlamydospores per kg BW were orally administered during seven days: (T1) non treated control group, (T2) 1 x 10(6), (T3) 2.5 x 10(6) and (T4) 5 x 10(6). Three lambs, infected with H. contortus, were used per group. Faeces were obtained from the rectum of each lamb during the fungal administration period (days 0-6) and for six days after that period. Four coproculture replicates were made from each animal in days 2, 4, 6, 8 and 10. A higher chlamydospore dose produced higher CPG in faeces (p < 0.05), but a clear dose dependent effect was not found either in the larvae reduction or in the CPG:EPG ratio. When ratios were re-analyzed, independently of the treatment groups of origin, a better efficacy was obtained with a ratio from 5 to 10 CPG:EPG and a higher ratio (> 10 per egg) showed a lower reduction efficacy (p < 0.05). The binomial analysis showed that for each unit of increment in CPG:EPG ratio there was a reduction of larvae number until a point (between 5 and 10 CPG:EPG) where no further reduction was detected. The surface response test indicated that the number of larvae was reduced by CPG until possible saturation. The highest CPG:EPG ratios did not necessarily improve efficacy of D. flagrans.

Comparing different maize supplementation strategies to improve resilience and resistance against gastrointestinal nematode infections in browsing goats.

The effect of maize grain supplementation on the resilience and resistance of browsing Criollo goat kids against gastrointestinal nematodes was evaluated. Five-month-old kids (n = 42), raised worm-free, were allocated to five groups: infected + not supplemented (I-NS; n = 10), infected + maize supplement at 108 g/d (I-S108; n = 8), maize supplement at 1% of body weight (BW) (I-S1%; n = 8), maize supplement at 1.5% BW (I-S1.5%; n = 8), or infected + supplemented (maize supplement 1.5% BW) + moxidectin (0.2 mg/kg BW subcutaneously every 28 d) (T-S1.5%; n = 8). Kids browsed daily (7 h) in a tropical forest for 112 days during the rainy season. Kids were weighed weekly to adjust supplementary feeding. Hematocrit (Ht), hemoglobin (Hb), and eggs per gram of feces were determined fortnightly. On day 112, five goat kids were slaughtered per group to determine worm burdens. Kids of the I-S1.5% group showed similar body-weight change, Ht and Hb, compared to kids without gastrointestinal nematodes (T-S1.5%), as well as lower eggs per gram of feces and Trichostrongylus colubriformis worm burden compared to the I-NS group (P > 0.05). Thus, among the supplement levels tested, increasing maize supplementation at 1.5% BW of kids was the best strategy to improve their resilience and resistance against natural gastrointestinal nematode infections under the conditions of forage from the tropical forest.