Bio-directed Chemical Study of Pleurotus ostreatus Spent Substrate and Its Nematicidal Activity.

PURPOSE: In the present study, the nematicidal activity of the chemical fractionation of the spent substrate of the edible mushroom Pleurotus ostreatus against eggs and L3 larvae of Haemonchus contortus was evaluated. METHODS: The hydroalcoholic extract of the spent substrate was subjected to a bipartition with ethyl acetate giving two fractions: one aqueous (F. Ac) and one organic (F. AcOET). Both fractions were evaluated against eggs and L3 larvae at different concentrations (5, 2.5, 1.25, 0.625 and 0.3125 mg/mL) and 2% methanol, PBS and thiabendazole (5 mg/mL) as controls. Chemical fractionation of F. AcOET was performed in open column chromatography where 76 fractions were obtained and when analyzed by thin layer chromatography (TLC) were grouped into 11 mixtures (R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11). These mixtures were evaluated at 10 mg/mL against eggs and L3 larvae. The data obtained were analyzed by ANOVA and a Tukey test in the SAS V9 program. RESULTS: The results showed that, in the evaluation of F. AcOET and F. Ac, for the inhibition of egg hatching, the highest percentages were 78.80 and 76.89% at 5 mg/mL, respectively. As for the percentage of larval mortality, F. AcOET obtained 60.91 and F. Ac 29.77% at 5 mg/mL. The results of the evaluations of the mixtures showed that mixtures R4, R5 obtained 100 and 95.41% larval mortality and mixtures R6 and R7 presented 100% inhibitory activity of egg hatching, so these were analyzed by gas chromatography mass spectrometry finding compounds such as vanillin, ß-sitosterol, ρ-methyl ρ-hydroxycinnamate and ρ-hydroxybenzaldehyde. CONCLUSION: The results of the present study demonstrate that the spent substrate of P. ostreatus has potential anthelmintic activity against H. contortus. Moreover, by reusing and taking advantage of this substrate, its environmental pollution effects can be reduced.

A Review of the Impact of Climate Change on the Epidemiology of Gastrointestinal Nematode Infections in Small Ruminants and Wildlife in Tropical Conditions.

Climate change is causing detrimental changes in living organisms, including pathogens. This review aimed to determine how climate change has impacted livestock system management, and consequently, what factors influenced the gastrointestinal nematodes epidemiology in small ruminants under tropical conditions. The latter is orientated to find out the possible solutions responding to climate change adverse effects. Climate factors that affect the patterns of transmission of gastrointestinal parasites of domesticated ruminants are reviewed. Climate change has modified the behavior of several animal species, including parasites. For this reason, new control methods are required for controlling parasitic infections in livestock animals. After a pertinent literature analysis, conclusions and perspectives of control are given.

Nematocidal activity of hydroalcoholic extracts of spent substrate of Pleurotus djamor on L3 larvae of Haemonchus contortus.

The objective of this study was to evaluate and compare the in vitro lethal effect of the hydroalcoholic extract of the spent substrate of Pleurotus djamor ECS-123, obtained at 15 days of colonization (SPS) and at the first (SPS1) and second (SPS2) harvests, against infective larvae L3 of Haemonchus contortus. The in vitro lethal effect was evaluated by the L3 larval mortality test (LM) using six concentrations: 1.25, 2.5, 5, 10, 20, and 40 mg/mL, with ivermectin and thiabendazole (5 mg/mL) as controls. The first harvest extract (SPS1) of strain ECS-123 was subjected to liquid-liquid bipartition, which resulted in two fractions: aqueous (PdAcO) and ethyl acetate (PdAct). The chemical fractionation of PdAct with the highest mortality rate (80.11 %) was carried out with open-column chromatography, giving a total of 13 fractions, which were analyzed by thin-layer chromatography (TLC) and grouped into 5 mixtures (R1;1-3, R2;4-7, R3;8-9, R4;10-11 and R5;12-13). Subsequently, the mixtures were evaluated against H. contortus L3 larvae. Finally, the components of the mixtures with the highest nematocidal effects were evaluated by gas chromatography coupled to mass spectrometry (GC-MS). The data were analyzed with a completely randomized design through ANOVA using the generalized linear model (GLM) with the "R" program. The purification and characterization of R4 and R5 by GC-MS revealed the presence of the following compounds: veratryl alcohol, 4-hydroxy-3,5,5 trimethyl-4-[3-oxo-1-butenyl]-2- cyclohexen-1-one, caffeine and 5,6-dimethoxy-1(3 H) isobenzofuranone. This information allowed for the identification of nematocidal compounds in the degraded substrate of P. djamor, an activity that had not been reported previously.

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.

The Possible Biotechnological Use of Edible Mushroom Bioproducts for Controlling Plant and Animal Parasitic Nematodes.

The present paper reviewed publications on the nematocidal activity of edible mushrooms (EM) and their potential use as sustainable tools for the control of parasitic nematodes affecting agriculture and livestock industry. Nematodes are organisms living in the soil and animals' guts where they may live as parasites severely affecting economically important crops and farm animals, thus causing economic losses to worldwide agriculture. Traditionally, parasitic nematodes have been controlled using commercial pesticides and anthelmintic (AH) drugs. Over the years, nematodes developed resistance to the AH drugs, reducing the usefulness of many commercial drugs. Also, the use of pesticides/anthelmintic drugs to control nematodes can have important negative impacts on the environment. Different EM have been not only used as food but also studied as alternative methods for controlling several diseases including parasitic nematodes. The present paper reviewed publications from the last decades about the nematocidal activity of EM and assessed their potential use as sustainable tools for the control of nematodes affecting agriculture and livestock industry. A reduced number of reports on the effect of EM against nematodes were found, and an even smaller number of reports regarding the potential AH activity of chemical compounds isolated from EM products were found. However, those studies have produced promising results that certainly deserve further investigation. It is concluded that EM, their fractions and extracts, and some compounds contained in them may have biotechnological application for the control of animal and plant parasitic nematodes.

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.

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.

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.

Effects of polyphenol removal methods on the in vitro exsheathment inhibitory activity of Lysiloma latisiliquum extracts against Haemonchus contortus larvae.

It has been suggested that the anthelmintic (AH) activity of tannin-rich forages is related to their tannin content. However, reports on tropical fodders such as Lysiloma latisiliquum describe the same AH activity after the addition of tannin-blocking agents, suggesting that the activity either depends on the method of tannin blocking/removal or is due to other type of secondary metabolites. This study compared both the effect of the drying process of the plant material and the effect of different polyphenol removal methods on the AH activity of L. latisiliquum acetone-water extracts when tested against Haemonchus contortus. The results showed that the extraction of oven-dried (OD) leaves of L. latisiliquum yielded a CT-free extract with high AH activity. However, polyphenol-free fractions showed similar or lower AH activity levels as of those original OD extract. HPLC analysis confirmed that common polyphenolic metabolites are not responsible for the AH activity of L. latisiliquum.