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.

Insecticidal activity of two organic extracts from Libidibia coriaria (Jacq.) Schltdl. fruits against Spodoptera frugiperda J. E. Smith.

This study describes the chemical characterisation of two extracts (ethyl acetate, EtOAc-E and hexanic, Hx-E) from Libidibia coriaria fruits and their insecticidal properties on Spodoptera frugiperda. The HPLC analysis in EtOAct-E revealed the presence of ellagic acid and phenolic compounds. The CG-MS analysis in Hx-E revealed the presence of Hexadecanoic acid, 11-Methylheptacosane, Dodecanoic acid and Nonacosane as major compounds. The application of both extracts was performed on the dorsal part of each larva via aspersion. The larval mortality, relative growth and emergence percentage of adults were evaluated. The Hx-E caused a 93.33% mortality at 100 mg/mL at 24 h post-application. A minor relative growth with both EtOAc-E (12.64 mg) and Hx-E (7.90 mg) was observed compared with their respective negative controls (methanol = 25.05 mg and tween20 = 24.53 mg). The lowest emergence percentage of adults with the Hx-E (25%) at 50 mg/mL was observed. Libidibia coriaria fruits exhibit insecticidal properties against S. frugiperda.

Assessing the In Vitro Individual and Combined Effect of Arthrobotrys oligospora and A. musiformis (Orbiliales) Liquid Culture Filtrates against Infective Larvae of the Sheep Blood-Feeding Nematode Haemonchus contortus (Trichostrongylidae).

BACKGROUND: Arthrobotrys species are nematophagous fungi that secrete extracellular nematocidal products (ECP). The individual and combined effects of ECP from Arthrobotrys oligospora (Ao) and A. musiformis (Am) growth in liquid media against Haemonchus contortus L3 (HcL3) were assessed. METHODS: The isolation, morphological (MI) and molecular identification (Mol-I), assessment of nematocidal activity (NA) of fungal liquid culture filtrates (LCF) in two liquid media alone and in combination and the myco-compound profile identification (MCP) were performed. RESULTS: The MI suggested that the fungi corresponded to the species Ao and Am. This result was confirmed by PCR analysis followed by sequencing, alignment and a phylogenetic analysis. Likewise, the highest Hc mortalities were 91.4% with individual LCF of Am and 86.2% with those of Ao at the highest concentration (100 mg/mL) in Czapek-Dox Broth. The combination of both LCF resulted in a similarly high larval mortality with no statistical differences in relation to individual activity (p > 0.05). The MCP showed the presence of alkaloids in both fungi. Coumarins, sterols and saponins were found only in Ao. MAIN CONCLUSIONS: Both fungi produced ECP with a high NA that could be identified and assessed in future studies as potential natural anthelmintic compounds.

In vitro ovicidal and larvicidal activity of a hydroalcoholic extract and its fractions from Cyrtocarpa procera fruits on Haemonchus contortus.

This study describes the in vitro anthelmintic effect of a hydroalcoholic extract (HA-E) and its fractions from Cyrtocarpa procera fruits against Haemonchus contortus eggs and infective larvae. The HA-E was subjected to bipartition using ethyl acetate, which resulted in an aqueous fraction (Aq-F) and an organic fraction (EtOAc-F). The HA-E and both fractions were tested using the egg hatching inhibition assay (EHIA) and the larval mortality test (LMT). Fractionation of the EtOAc-F was achieved using different chromatographic processes, i.e., open glass column and HPLC analysis. Fractionation of the EtOAc-F gave 18 subfractions (C1R1-C1R18), and those that showed the highest yields (C1R15, C1R16, C1R17 and C1R18) were subjected to anthelmintic assays. The HA-E and the EtOAc-F displayed 100% egg hatching inhibition at 3 and 1 mg/mL, respectively, whereas Aq-F exhibited 92.57% EHI at 3 mg/mL. All subfractions tested showed ovicidal effect. Regarding the larval mortality test, HA-E and EtOAc-F exhibited a larvicidal effect higher than 50% at 50 and 30 mg/mL, respectively. The subfractions that showed the highest larval mortality against H. contortus were C1R15 and C1R17, with larval mortalities of 53.57% and 60.23% at 10 mg/mL, respectively. Chemical analysis of these bioactive subfractions (C1R15 and C1R17) revealed the presence of gallic acid, protocatechuic acid, and ellagic acid. This study shows evidence about the ovicidal and larvicidal properties of C. procera fruits that could make these plant products to be considered as a natural potential anthelmintic agents for controlling haemonchosis in goats and sheep.

Synergistic Interaction Effect of Artemisia cina n-hexane Extract and Tagetes lucida Ethyl Acetate Extract on Haemonchus Contortus.

PURPOSE: We analysed the possible synergistic activity among active extracts from Artemisia cina and Tagetes lucida combinations on Haemonchus contortus, a nematode parasitising sheep. METHODS: The work was carried out in vitro on eggs and infective larvae (L3) of H. contortus. The results were analysed with SAS 9.1, applying the ANOVA and Tukey test, and the lethal concentration (LC) values LC50 and LC90 were determined with regression analysis, employing Proc Probit of SAS 9.1. Additionally, the lethal concentration (LC) was calculated with LC50 and LC90 to determine the synergistic effect. RESULTS: The results demonstrated a high efficacy of the two plants studied on both nematode eggs and L3 larvae as well as of their combinations. The highest egg hatching inhibition was obtained with a 50/50 combination, and the best larvae mortality was obtained with 25% A. cina and 75% T. lucida at 10 mg/mL. Additionally, this combination showed a synergistic effect. CONCLUSION: The two plant species studied here can be applied as natural anthelmintic alternatives due to their high bioactive effect and synergistic response.

Anthelmintic efficacy of an organic fraction from Guazuma ulmifolia leaves and evaluation of reactive oxidative stress on Haemonchus contortus.

This study describes the anthelmintic efficacy of an organic fraction (EtOAc-F) from Guazuma ulmifolia leaves and the evaluation of its reactive oxidative stress on Haemonchus contortus. The first step was to assess the anthelmintic effect of EtOAc-F at 0.0, 3.5, 7.0 and 14 mg kg of body weight (BW) in gerbil's (Meriones unguiculatus) artificially infected with H. contortus infective larvae (L3). The second step was to evaluate the preliminary toxicity after oral administration of the EtOAc-F in gerbils. Finally, the third step was to determine the relative expression of biomarkers such as glutathione (GPx), catalase (CAT), and superoxide dismutase (SOD) against H. contortus L3 post-exposition to EtOAc-F. Additionally, the less-polar compounds of EtOAc-F were identified by gas mass spectrophotometry (GC-MS). The highest anthelmintic efficacy (97.34%) of the organic fraction was found in the gerbils treated with the 14 mg/kg of BW. Histopathological analysis did not reveal changes in tissues. The relative expression reflects overexpression of GPx (p<0.05, fold change: 14.35) and over expression of SOD (p≤0.05, fold change: 0.18) in H. contortus L3 exposed to 97.44 mg/mL of EtOAc-F compared with negative control. The GC-MS analysis revealed the presence of 4-hydroxybenzaldehyde (1), leucoanthocyanidin derivative (2), coniferyl alcohol (3), ferulic acid methyl ester acetate (4), 2,3,4-trimethoxycinnamic acid (5) and epiyangambin (6) as major compounds. According to these results, the EtOAc-F from G. ulmifolia leaves exhibit anthelmintic effect and increased the stress biomarkers on H. contortus.

First Record of Flavocillium subprimulinum (Cordycipitaceae, Hypocreales) in Mexico: Morphological and Molecular Characterisation, Nematocidal Activity of Its Liquid Culture Filtrates against Haemonchus contortus and Protease Activity.

This is the first record of the fungus Flavocillium subprimulinum in Mexico. The isolate was taxonomically characterised and cultured in potato dextrose broth (PDB), Czapek-Dox broth (CzDoxB), and sweet potato dextrose broth (SPDB) to obtain its filtrates (FLCF). The nematocidal activity (NA) of three FLCF concentrations was assessed against Haemonchus contortus L3. Protease activity (PA) was assessed with SDS-PAGE, followed by a zymogram. The NA of the FLCF reached 94.43% in PDB and 95.82% in CzDoxB, respectively, at 100 mg/mL. Lower mortality (64%) was found in SPDB at 100 mg/mL. SDS-PAGE showed bands (in PBS) of ~25, ~40, and ~55 kDa. The zymogram showed protein bands (PBs) with PA in the media, including PBs of ~14, ~40, and ~55 kDa. This study establishes the basis for exploring the potential use of this fungus against H. contortus, which is considered the most pathogenic parasite affecting lambs.

Phenolic compounds in the fruits of Enterolobium cyclocarpum and the inhibition of Haemonchus contortus eggs.

The fabacean Enterolobium cyclocarpum is a tree with multiple uses in production systems and its fruits serve as food for livestock, in addition, they contain phenolic compounds. The ovicidal effect of the secondary compounds of the Enterolobium cyclocarpum fruits extracted with hydroalcoholic solvent (HA-E) and the aqueous (Aq-F) and organic (AcOEt-F) fractions was evaluated. Additionally, a phytochemical analysis of the extract and fractions was performed by high performance liquid chromatography (HPLC). The HA-E showed an ovicidal effect close to 100% with the concentration evaluated of the 100 mg/mL. The fractionation of the extract allowed to potentiate the activity in the Aq (94.05% at 12 mg/mL) and AcOEt (99.45% at 3 mg/mL) fractions. The secondary compounds extracted from the fruits of E. cyclocarpum in the HA-E and fractions were flavonols, coumaric and ferulic acids and other derivatives of hydroxycinnamic acid and were responsible for the ovicidal activity observed against H. contortus.

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.

Bioactive compounds from the parasitic plant Arceuthobium vaginatum inhibit Haemonchus contortus egg hatching.

The aim of this study was to assess the in vitro ovicidal activity of an ethyl acetate extract from Arceuthobium vaginatum (EtOAc-E) and their subfractions (AvR5-AvR14) against Haemonchus contortus using the egg hatching inhibition (EHI) test. The EtOAc-E and subfractions were tested at 0.12-2.00 and at 0.015-2.0 mg/mL, respectively. Distilled water and methanol (2%) were used as negative controls and Thiabendazole (0.10 mg/mL) as a positive control. Treatments with a dependent effect on concentration were subjected to regression analysis to determine the effective concentrations (EC50 and EC90). The major secondary compounds present in the extract and subfractions were identified by high performance liquid chromatography (HPLC). The EtOAc-E and AvR9 exhibited the best ovicidal effect recording 97.5 and 100% of EHI at 0.25 mg/mL, respectively. The EtOAc-E and AvR9 displayed an EC50= 0.12 and 0.08 mg/mL, respectively. The HPLC analysis in the EtOAc-E and bioactive fractions indicated the presence of a polyphenol, glycosylated flavanones, quercetin glucoside, cinnamates, coumarin, cinnamic acid derivative, ferulic acid, coumarate, naringenin, protocatechuic acid and naringin. Results demonstrated that A. vaginatum extract and fraction is able to inhibit the egg hatch process of H. contortus and could be a viable option for the control of small ruminant haemonchosis.

Study of a Mexican isolate of Arthrobotrys musiformis (Orbiliales): Predatory behavior and nematocidal activity of liquid culture filtrates against Haemonchus contortus (Trichostrongylidae), protein profile and myco-constituent groups.

A Mexican isolate of the nematophagous fungus Arthrobotrys musiformis was obtained from a soil sample from the Chapultepec ecological reserve zone, in Cuernavaca, Morelos, Mexico. This isolate demonstrated an important predatory activity (74.9%) against the parasitic nematode Haemonchus contortus (L3) and its fungal liquid culture filtrates (LCF) grown in two media showed the following highest nematocidal activities (NA): In Czapek-DoxBroth (CzDoxB) 80.66% and potato-dextrose broth (PDB) 49.84%. Additionally, two major compounds derived from carboxylic acids and two derivates from alkane group were identified by GC-MS. These compounds have been associated to many biological activities. On the other hand, the protein profile analysis by SDS-electrophoresis followed by a zymogram revealed a 10 kDa protein with protease activity. This study provides important information for future experiments focused to explore the potential use of this protein as well as the identified bioactive compounds presents in the LCF as potential candidates against sheep haemonchosis.

Recent Advances in the Control of Endoparasites in Ruminants from a Sustainable Perspective.

Consumer awareness of animal welfare and environmental health has led to a plateau level of global consumption putting serious pressure on the livestock industry [...].

In vitro ovicidal effect of p-coumaric acid from Acacia bilimekii aerial parts against Haemonchus contortus.

Acacia bilimekii is a plant with a high content of protein, fibre, and condensed tannins, making it an excellent feed for small ruminants with anthelmintic potential. This study aimed to evaluate the ovicidal activity of a hydroalcoholic extract (Ab-HA) and fractions from A. bilimekii aerial parts on Haemonchus contortus. The ovicidal activity of the Ab-HA extract and its fractions obtained by chromatographic fractionation were evaluated through the egg hatching inhibition (EHI) test. The results showed that the Ab-HA extract had 91% EHI at 20,000 µg/mL with a mean effective concentration (EC50) of 9260 µg/mL. After liquid-liquid fractionation of Ab-HA extract, the aqueous fraction (Ab-Aq) did not show ovicidal activity, whereas the organic fraction (Ab-EtOAc) showed a better EHI than the Ab-HA extract (98.9% at 2500 µg/mL). Then, the chemical fractionation of Ab-EtOAc allowed obtaining six bioactive fractions (AbR12-17) with an EHI greater than 90% at 1500 µg/mL. The best treatment was AbR15 (98.7% EHI at 750 µg/mL). Chemical analysis by HPLC-PDA of AbR15 indicated the presence of p-coumaric acid and the flavone luteolin as major compounds. Additionally, the commercial p-coumaric acid standard was evaluated in the EHI assay and showed an EHI of 97% at 62.5 µg/mL. Meanwhile, the confocal laser scanning microscopy analysis demonstrated a colocalization effect between p-coumaric acid and the H. contortus embryonated eggs. These results indicate that due to their major chemical compounds (including p-coumaric acid), the aerial parts of the plant A. bilimekii, could be considered as natural potential tool for controlling haemonchosis in small ruminants.

Assembly and Analysis of Haemonchus contortus Transcriptome as a Tool for the Knowledge of Ivermectin Resistance Mechanisms.

Haemonchus contortus (Hc) is an important parasitic nematode of small ruminants. In this study we assembled the transcriptome of Hc as a model to contribute to the knowledge about the profile of the differential gene expression between two Mexican Hc strains under different anthelmintic resistance statuses, one susceptible and the other resistant to ivermectin (IVMs and IVMr, respectively), in order to improve and/or to have new strategies of control and diagnosis. The transcript sequence reads were assembled and annotated. Overall, ~127 Mbp were assembled and distributed into 77,422 transcript sequences, and 4394 transcripts of the de novo transcriptome were matched base on at least one of the following criteria: (1) Phylum Nemathelminthes and Platyhelminthes, important for animal health care, and (2) ≥55% of sequence identity with other organisms. The gene ontology (GO) enrichment analysis (GOEA) was performed to study the level of gene regulation to IVMr and IVMs strains using Log Fold Change (LFC) filtering values ≥ 1 and ≥ 2. The upregulated-displayed genes obtained via GOEA were: 1993 (for LFC ≥ 1) and 1241 (for LFC ≥ 2) in IVMr and 1929 (for LFC ≥ 1) and 835 (for LFC ≥ 2) in IVMs. The enriched GO terms upregulated per category identified the intracellular structure, intracellular membrane-bounded organelle and integral component of the cell membrane as some principal cellular components. Meanwhile, efflux transmembrane transporter activity, ABC-type xenobiotic transporter activity and ATPase-coupled transmembrane transporter activity were associated with molecular function. Responses to nematicide activity, pharyngeal pumping and positive regulation of synaptic assembly were classified as biological processes that might be involved in events related to the anthelmintic resistance (AR) and nematode biology. The filtering analysis of both LFC values showed similar genes related to AR. This study deepens our knowledge about the mechanisms behind the processes of H. contortus in order to help in tool production and to facilitate the reduction of AR and promote the development of other control strategies, such as anthelmintic drug targets and vaccines.

3'-Demethoxy-6-O-Demethylisoguaiacin and Norisoguaiacin Nematocidal Lignans from Artemisia cina against Haemonchus contortus Infective Larvae.

Artemisia cina is a plant used in traditional Chinese medicine as a remedy for parasitic diseases. This study describes the isolation and chemical characterization of anthelmintic compounds of A. cina against Haemonchus contortus infective larvae (L3) through lethal testing. Previously, three extracts-n-hexane (HexAc), ethyl acetate (EtOAc) and methanol (MeOAc)-were evaluated at concentrations of 4 to 0.5 mg/mL, resulting in the HexAc extract with the greatest effect of 76.6% mortality of the larvae at 4 mg/mL. Then, this was chemically fractioned by polarity, obtaining seven fractions (C1F1-C1F7), and, when evaluated at concentrations from 2 to 0.25 mg/mL, the 2 mg/mL C1F5 fraction produced an effect against the nematode H. contortus of 100% mortality of the larvae. Thus, this fraction was fractionated again by column chromatography, obtaining twelve subfractions (C2F1-C2F12) which were evaluated from 1 to 0.125 mg/mL, with the C2F5 subfraction causing a nematicidal effect of 100% mortality. NMR analysis of one (1H, 13C and DEPT) and two dimensions (COSY, HSQC and HMBC) and mass spectrometry of this fraction allowed us to identify the mixture of 3'-demethoxy-6-O-demethylisoguaiacin and norisoguaiacin. Therefore, it can be assumed that the mixture of these compounds is responsible for the anthelmintic effect. These results indicate that A. cina containing anthelmintic compounds and might be used as an antiparasitic drug against H. contortus.

Coffee Pulp: A Natural Alternative for Control of Resistant Nematodes in Small Ruminants.

Goat production in Mexico is an important economic activity that is affected by different gastrointestinal nematode (GIN) species. GINs resistant to commercial anthelmintics have been reported. Plant extracts or agro-industrial by-products, such as coffee pulp, have been proposed as control alternatives, given their secondary metabolite content. The aim of the present study was to determine the anthelmintic activity of the hydroalcoholic extract of coffee pulp against benzimidazole-resistant GINs. Stool samples were collected from goats, from which GIN eggs were identified and quantified. Molecular techniques confirmed the genus of GINs and their benzimidazole resistance profile. The percentage of egg hatching inhibition (% EHI) and larval mortality (% LM) with the hydroalcoholic extract of coffee pulp was determined at concentrations from 200 to 0.39 mg/mL. The genera Haemonchus spp. and Trichostrongylus spp. were identified, and the presence of the ß-tubulin gene mutation, associated with benzimidazole (BZ) resistance, was determined. Hydroalcoholic extract of coffee pulp inhibited 100% of egg hatching at 200 and 100 mg/mL, with no larvicidal effect at the evaluated concentrations.

In vitro and in vivo anthelmintic properties of Caesalpinia coriaria fruits against Haemonchus contortus.

The purpose of the present study was to assay the in vitro and in vivo anthelmintic activity (AA) of Caesalpinia coriaria (Cc) mature fruits against the nematode Haemonchus contortus (Hc). The Hc infective larvae were used to assess the in vitro AA through larval mortality assay. The exposure of larvae to the different treatments was performed in 96-well microtitration plates. The treatments were as follows: hydroalcoholic extract (HA-E, at 25-100 mg/mL), aqueous fraction (Aq-F, at 12.5-50 mg/mL), organic fraction (EtOAc-F at 12.5-50 mg/mL), compounds (1, methyl gallate and 2, gallic acid at 1.25-10 mg/mL), positive control (ivermectin at 5 mg/mL) and two negative controls (distilled water and 4% methanol). After exposure, dead and live larvae were quantified and results were compared to their controls. The in vivo assay was carried out by a faecal egg count reduction test (FECRT); artificially infected goat kids (F1: Boer x Nubia) were treated with Cc ground dried fruits to assess the AA. The treatments were established as follows: G1-untreated goats (negative control), G2-goats dewormed with ivermectin (positive control), G3-goats fed with Cc mature fruits (10% of their diet). Results in both in vitro and in vivo assays were analysed using an ANOVA through random design, applying a general linear model and mixed models. The in vitro results showed an evident larvicidal effect of the HA-E, EtOAc-F from Cc, indicating that the compound responsible for the AA was gallic acid. The results of the in vivo study corroborated the anthelmintic properties of Cc, reaching 78.6% reduction in the elimination of Hc eggs per gram of faeces. This plant represents a potential natural anthelmintic for the control of haemonchosis in goats under grazing conditions. Future studies should standardise the Cc extract or dried fruits for use in the management of nematodiasis in goat herds.

Phenolic Acids and Flavonoids from Pithecellobium dulce (Robx.) Benth Leaves Exhibit Ovicidal Activity against Haemonchus contortus.

Pithecellobium dulce (Robx.) Benth is an arboreal legume used in traditional medicine for the treatment of several ailments, including a number of intestinal disorders, and as a natural deworming. The objective of this study was to evaluate the ovicidal activity of a hydroalcoholic extract (HA-E) and its fractions (aqueous, Aq-F and organic, EtOAc-F) from P. dulce leaves, as well as subfractions (C1F1-C17) obtained from EtOAc-F against Haemonchus contortus eggs. The HA-E, Aq-F, and EtOAc-F were assessed at 0.03-5.00 mg/mL and subfractions (C1F1-C17) were assessed at 0.06-1.00 mg/mL. The HA-E and Aq-F showed an ovicidal activity close to 100% at 2.5 mg/mL, and EtOAc-F displayed the highest anthelmintic effect (100% at 0.25 mg/mL). Meanwhile, the sub-fractions with the highest ovicidal effect were C1F6, C1F9, C1F10, C1F11, and CIF13. The main compounds identified in the most active fractions (C1F9, C1F11, and C1F13) were kaempferol (1), quercetin (2), coumaric acid (3), ferulic acid (4), luteolin 7-O-rhamnoside (5), quercetin 3-O-rhamnoside (6), and a caffeoyl derivate (NI). The results indicate that P. dulce leaves exhibit a potent anthelmintic activity and contain bioactive compounds able to inhibit egg hatching in H. contortus. Therefore, this plant could be used for the control of gastrointestinal nematodes in small ruminants.

Arthrobotrys musiformis (Orbiliales) Kills Haemonchus contortus Infective Larvae (Trichostronylidae) through Its Predatory Activity and Its Fungal Culture Filtrates.

Haemonchus contortus (Hc) is a parasite affecting small ruminants worldwide. Arthrobotrys musiformis (Am) is a nematode-trapping fungi that captures, destroys and feeds on nematodes. This study assessed the predatory activity (PA) and nematocidal activity (NA) of liquid culture filtrates (LCF) of Am against Hc infective larvae (L3), and additionally, the mycochemical profile (MP) was performed. Fungal identification was achieved by traditional and molecular procedures. The PA of Am against HcL3 was performed in water agar plates. Means of non-predated larvae were recorded and compared with a control group without fungi. LCF/HcL3 interaction was performed using micro-tittering plates. Two media, Czapek−Dox broth (CDB) and sweet potato dextrose broth (SPDB) and three concentrations, were assessed. Lectures were performed after 48 h interaction. The means of alive and dead larvae were recorded and compared with proper negative controls. The PA assessment revealed 71.54% larval reduction (p < 0.01). The highest NA of LCF was found in CDB: 93.42, 73.02 and 51.61%, at 100, 50 and 25 mg/mL, respectively (p < 0.05). Alkaloids and saponins were identified in both media; meanwhile, coumarins were only identified in CDB. The NA was only found in CDB, but not in SPDB. Coumarins could be responsible for the NA.

In Vitro Anthelmintic Activity of a Hydroalcoholic Extract from Guazuma ulmifolia Leaves against Haemonchus contortus.

The purpose of the present study was to assess the ovicidal and larvicidal activity of a hydroalcoholic extract (HAE) and their fractions (aqueous, Aq-F and organic, EtOAc-F) from Guazuma ulmifolia leaves using Haemonchus contortus as a biological model. The egg hatching inhibition (EHI) and larval mortality against infective larvae (L3) tests were used to determine the anthelmintic effect of the treatments. The extract and fractions were tested at different concentrations against eggs and L3. Additionally, distilled water and methanol were used as negative controls and ivermectin as a positive control. The extract and fractions were subjected to HPLC analysis to identify the major compounds. The HAE displayed the highest ovicidal activity (100% EHI at 10 mg/mL). Fractionation of the HA extract allowed increasing the nematicidal effect in the EtOAc-F (100% EHI at 0.62 mg/mL and 85.35% mortality at 25 mg/mL). The phytochemical analysis of the extract and fractions revealed the presence of kaempferol, ethyl ferulate, ethyl coumarate, flavonol, luteolin, ferulic acid, luteolin rhamnoside, apigenin rutinoside, coumaric acid derivative, luteolin glucoside, and quercetin glucoside. These results suggest that G. ulmifolia leaves could be potential candidates for the control of H. contortus or other gastrointestinal parasitic nematodes.