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.

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.

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.

In Vitro Nematocidal Properties from Two Extracts: Lippia graveolens Leaves and Delonix regia Flowers Against Eggs and Infective Larvae of Haemonchus contortus.

The objective of this study was to assay the ovicidal and larvicidal effects of two hydroalcoholic extracts (HA-E) and their aqueous fractions (Aq-F) and organic fractions (EtOAc-F) from Lippia graveolens (leaves) and Delonix regia (flowers) against the parasitic nematode Haemonchus contortus. The egg hatching inhibition test (% EHI) and mortality of infective larvae (% mortality) were used as biological models. The treatments of each plant (mg/mL, % EHI, and % mortality) were assigned as follows: HA-E (12.5-50, EHI; 25-200, mortality), Aq-F (6.25-25.00, EHI; 12.5-50, mortality), EtOAc-F (0.37-25, EHI; 3.12-50, mortality), negative controls (distilled water and methanol at 2% and 4%), and positive control (ivermectin at 5 mg/mL). Data were analyzed using a completely randomized design through an analysis of variance, considering the EHI and mortality percentages as the response variables. To identify the secondary metabolites, both plants were subjected to qualitative chemical analysis and HPLC. The results showed that both plants exhibited highly ovicidal and larvicidal effects. The bipartition in each HA extract allowed the increase of the anthelmintic effect in EtOAc-F, achieving a total ovicidal activity at 1.56 mg/mL and a larvicidal effect higher than 90% at 25 mg/mL. The qualitative chemical profile of both vegetal species identified alkaloids, flavonoids, condensed tannins, terpenes, and saponins. These results suggest that either L. graveolens leaves or D. regia flowers could be potential candidates for the control of parasitic gastrointestinal nematodes in small ruminants. However, in vivo studies with experimental animals to evaluate their possible toxicity and anthelmintic effect are necessary.

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.

Oxalis tetraphylla (Class: Magnoliopsidae) Possess Flavonoid Phytoconstituents with Nematocidal Activity against Haemonchus contortus.

The nematocidal activity of an Oxalis tetraphylla hydroalcoholic extract against the nematode Haemonchus contortus (Hc) was assessed in vitro and the major compounds associated with nematocidal activity were identified. One hydroalcoholic extract was obtained from O. tetraphylla stems and leaves (Ot HE-SLE). The in vitro lethal concentrations (LC50 and LC90) against both eggs and exsheathed Hc infective larvae (L3) were assessed. Ot HE-SLE showed a potent ovicidal activity (LC50 = 0.213 mg/mL; LC90 = 0.71 mg/mL) and larvicidal effect (LC50 = 28.01 mg/mL; LC90 = 69.3 mg/mL). Later on, the extract was bipartitioned to obtain an ethyl acetate phase (EtOAc-Ph) and an aqueous phase (Aq-Ph). Both phases were assessed against Hc eggs at 0.25 and 1.0 mg/mL concentrations. The results with EtOAc-Ph showed 93.6% ovicidal activity, while 96.6% was recorded with Aq-Ph at 48 h post-confrontation (PC). In the case of larvicidal activity, both phases were assessed at 28 mg/mL; Aq-Ph showed >80% larvicidal activity 24 and 72 h PC, while EtOAc-Ph did not show important activity. HPLC analysis showed the presence of coumaric acid and flavonols. Flavonol compounds were the major compounds and were associated with the nematocidal activity. Additionally, the Aq-Ph that showed the highest activity was purified, and the fraction F3 showed the highest nematocidal activity.

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.

Isorhamnetin: A Nematocidal Flavonoid from Prosopis Laevigata Leaves Against Haemonchus Contortus Eggs and Larvae.

Haemonchus contortus affect small ruminants all over the world. Anthelmintics cause resistance, contamination, and a risk of public health. Prosopis laevigata is a plant used as a home remedy against many diseases in Mexico. This study arose from a preliminary study where a P. laevigata hydroalcoholic extract (Pl-hae) showed anthelmintic activity (aa) against H. contortus. Searching for bioactive compounds (bac) with high aa, the Pl-hae was fractioned obtaining an aqueous (Aq-F) and an ethyl acetate fraction (EtAc-F), and a flavonoid with aa identified as isorhamnetin was obtained from EtAc-F. Both fractions were in vitro assessed by the egg hatch test (eht) and larval mortality (lm) assays. The bac obtained from EtAc-F were characterised by NMR analysis. The highest aa were recorded with EtAc-F, resulting in 100% eht and 80.45% lm at 0.75 and 30 mg/mL, respectively. Alterations in eggs and larvae attributed to isorhamnetin were recorded by environmental scanning electron microscopy, confocal laser scanning and by high-resolution digital-coupled camera. This flavonoid caused 100% eht at 0.07 mg/mL after 48 h and 100% lm at 7.5 mg/mL after 72 h exposure. Isorhamnetin has promising potential as an anthelmintic against sheep haemonchosis.