Lecanicillium psalliotae (Hypocreales: Cordycipitaceae) Exerts Ovicidal and Larvicidal Effects against the Sheep Blood-Feeding Nematode Haemonchus contortus through Its Liquid Culture Filtrates.

Nematophagous fungi (NF) form part of the soil microbiota and are natural enemies of nematodes, helping to regulate nematode populations. A verticillate NF isolated from soil from Tepalcingo, Mexico, was morphologically and molecularly characterised. This fungus was cultured in two different liquid media-Czapek-Dox broth (CzDoxB) and sweet potato dextrose broth (SPDB)-for 21 days. The ovicidal (OA) and larvicidal (LA) activities of fungal liquid culture filtrates (LCFs) were assessed in 96-well microtitre plates at different concentrations against Haemonchus contortus after 48 h. The morphological and molecular identification revealed the presence of Lecanicillium psalliotae. Additionally, the groups of compounds associated with nematocidal activity were determined from a qualitative chemical profile (QCP) using different reagents. The highest OA of the LCFs was obtained at 25 mg/mL from SPDB and CzDoxB and amounted to 97.2 and 99.06%, respectively. Meanwhile, the highest LA recorded with these LCFs at 100 mg/mL was 54.27% and 96.8%, respectively. The QCP revealed the presence of alkaloids and tannins in both LCFs that have previously been associated with nematocidal activity. Lecanicillium psalliotae exerted an important effect on H. contortus and could be of significance in future studies focused on the control and prevention of haemonchosis in small ruminants.

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.

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 [...].

Anthelmintic-Like Activity and Ultrastructure Changes Produced by Two Polyphenolic Combinations against Cooperia punctata Adult Worms and Infective Larvae.

Cooperia punctata is one of the most prevalent gastrointestinal nematodes affecting cattle under grazing conditions, and the increasing reports of anthelmintic resistance forces researchers to look for novel control measures. Previous reports have proposed the use of polyphenolic compound (PC) combinations (Coumarin:Quercetin (CuQ) and Caffeic-acid:Rutin (CaR)) against free-living stages (L3) of C. punctata. The objective of this study was to assess the in vitro motility inhibition of C. punctata adult worms and infective larvae using the Larval Motility Inhibition Assay (LMIA) and Adult Motility Inhibition Assay (AMIA), and to assess the structural and ultrastructural changes induced by both treatments using Scanning and Transmission Electron Microscopy. For the LMIA, infective larvae were incubated for 3 h in 0.8 mg mL-1 and 0.84 mg mL-1 of CuQ and CaR, respectively. For AMIA, six concentrations and five incubation periods (2, 4, 6, 12 and 24 h) were assessed using each PC combination. Cooperia punctata motility was calculated as a percentage and corrected using control motility percentages. A multiple comparisons Brown-Forsythe and Welch ANOVA test was used to compare larval motility; and to fit the dose-response in AMIA, data were analyzed with a non-linear regression four-parameter logistic equation with a variable slope, using the computer program GraphPad Prism® V.9.2.0. Although larval motility was barely affected by both treatments (p > 0.05), adult worm motility was inhibited 100% and 86.9% after 24 h incubation with CuQ and CaR, respectively (p < 0.05). The best fit EC50 for adult worm motility inhibition were 0.073 ± 0.071 mg mL-1 and 0.051 ± 0.164 mg mL-1 for CuQ and CaR, respectively. Main structural and ultrastructural lesions observed in both biological stages were: (i) L3 sheath-cuticle complex disruption, (ii) collagen fibers degradation; (iii) hypodermic detachment, (iv) seam cell apoptosis and (v) mitochondrial swelling. The alterations observed suggest that the PC combinations interfere with the anatomy and physiology of the locomotive apparatus of the nematodes.

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.

The immune gene expression induced by Cooperia punctata in naturally infected calves with resistance and susceptible phenotype traits.

The analysis of the immune gene expression was performed in Zebu × Holstein calves with resistant and susceptible phenotypes naturally infected with Cooperia punctata. Fourteen calves of 4 months old were grazed for 11 weeks under a tropical climate. The parasitic infection showed an average epg value of 1055 ± 1155 and an IgG optical density of 0.814 ± 0.0.037 with statistic differences among the different weeks (p < 0.05), and a pcv value of 24 ± 2.0 % (p > 0.05). High variation in epg value was observed, between 7 ± 7.14 and 4657 ± 1886, and, based on these differences; the infected hosts were classified as five resistant calves with epg ≤ 200 and nine susceptible calves with epg ≥ 300. Moreover, IgG levels displayed statistical differences between resistance and susceptible calves to C. punctata infection. The immune gene expression was analysed in three resistant and susceptible calves, respectively. Nine cytokine genes and the FCεR1A receptor were analysed at the 3rd and 11th weeks post-infection. In the first period upregulation was found, from 2.19- to 9.45-fold, (p < 0.05) for IL-2, -5, - 6, -10, TGF-ß and FCεR1A in the resistant group; the expression was decreased at the 11th week with low level of IgG. In contrast, downregulation for susceptible calves was found for nine immune genes and upregulation for INF-γ in both periods together with increased IgG levels. In conclusion, immune gene expression was regulated at the begging infection of C. punctata in resistant grazing calves. In contrast, suppression of important genes was involved in calves susceptible to C. punctata.

Effect of Gliricidia sepium leaves intake on larval establishment of Cooperia punctata in calves and bio-guided fractionation of bioactive molecules.

The aims of this study were: 1) to assess the anthelmintic effect of Gliricidia sepium on the establishment of C. punctata third-stage larvae (L3) in calves, and 2) to isolate and to elucidate an anti-exsheathment phytochemical from the plant offered during the trial. Twelve ¾ Holstein × Zebu calves were divided in two experimental groups: control (T1) and treatment (T2) (n = 6). After adaptation, each calf was infected with an oral dose of 400 C. punctata L3/Kg LW. Basal diet consisted of Digitaria decumbens hay (6.27% CP) and commercial concentrate (12% CP). In addition, during the experimental period T2 received fresh G. sepium leaves (26.88% CP) ad libitum. On day 9 post-infection, three calves per treatment were randomly selected for slaughter, and worm counts were performed. Larval establishment rates obtained were 13.44 ±â€¯0.13% and 3.1 ±â€¯1.42% for T1 and T2, respectively (P < .05). The reduction of larval establishment was 76.9%. The total length of worms recovered from the animals was also affected by the intake of G. sepium (P < .05). Phytochemicals present in G. sepium leaves offered to calves were isolated through silica gel columns and elucidated through Magnetic Nuclear Resonance (1H and 13C). Bio-guided isolation procedures lead to the elucidation of Oxytroside (Kaempferol 3-O-rhamnopyranosyl-(1 → 6)-ß-D-glucopyranoside-7-O-rhamnopyranoside), which fully inhibited the C. punctata exsheathment process (2400 µg mL-1). Gliricidia sepium represents an alternative to prevent severe C. punctata infections by reducing larval establishment in cattle.

In vitro larvicidal effect of a hydroalcoholic extract from Acacia cochliacantha leaf against ruminant parasitic nematodes.

The aim of this study was to evaluate the in vitro lethal effect of a hydroalcoholic extract (HAE) from Acacia cochliacantha leaf against three gastrointestinal nematodes species (Haemonchus contortus, H. placei and Cooperia punctata) of domestic ruminants. The HAE was assessed using five concentrations: 100, 125, 175, 150 and 200 mg/ml; 0.5% Ivermectin was used as a positive control and distilled water, as negative control. The data were normalized using the square root and analysed with a completely randomized design through ANOVA analysis using the general lineal model (GLM) of the SAS program. The HAE tannin content was determined through spectrophotometry (UV-visible) and the other major phenols, were identified by chromatographic processes. The results showed an in vitro larvicidal activity of the HAE against the three assessed nematode species with all assessed concentrations. A clear HAE increased concentration dependence effect was observed. The highest activity of the HAE was obtained at the highest concentration (close to 100%, P < 0.05). This result was similar to the one obtained with Ivermectin. On the other hand, the chemical analysis of HAE showed the presence of tannins, caffeoyls and coumaroyl derivates and quercetin as the main compounds. The results suggest that the HAE from this plant species possess in vitro anthelmintic properties. The identified compounds in this study would good candidates for further in vivo researches.

Actividad ovicida de extractos de cuatro especies de plantas contra el nematodo gastrointestinal Cooperia punctata / Ovicide activity of extracts of four plant species against the gastrointestinal nematode Cooperia punctata

El uso de plantas bioactivas representa una alternativa para el control de Cooperia punctata en el ganado de pastoreo. Los objetivos de este estudio fueron: (1) evaluar la actividad ovicida de extractos de cuatro especies de plantas contra C. punctata, (2) determinar la participación de los compuestos polifenólicos (cp) en la actividad antihelmíntica (ah) y (3) evaluar el mejor método de extracción para alcanzar una actividad ovicida. El bioensayo de inhibición de la eclosión de huevos (ieh) fue utilizado para evaluar el efecto ovicida de Leucaena leucocephala, Gliricidia sepium, Guazuma ulmifolia y Cratylia argentea; con diferentes métodos de extracción: acuoso (aq), acetona: agua (aw) y acetónico (ac). Los huevos de C. punctata fueron incubados por cuadruplicado a 0.6, 1.2, 2.4, 4.8 y 9.6 mg mL-1 de cada uno de los extractos de plantas. La participación de los cp fue determinada mediante el uso de polietilenglicol (peg). Los 12 extractos de plantas inhibieron la eclosión de huevos de modo dosis-dependiente, y las mejores concentraciones letales medias (CL50) fueron: 1.03 ± 0.17 para G. sepium-ac, y 7.90 ± 1.19 mg mL-1 para L. leucocephala-aq. Se observaron diferencias en la actividad ah según el método de extracción (P < 0.05). A mayor concentración, L. leucocephala-aq inhibió más del 50 % de la eclosión de C. punctata, entretanto, el extracto G. sepium-ac inhibió la eclosión en su totalidad. La adición de peg demostró la participación de los cp en la actividad ah de la mayoría de los extractos; no obstante, con G. sepium-ac, los cp no fueron los principales compuestos bioactivos. En general, el mejor método de extracción fue acetona:agua, pues permitió obtener una buena actividad ovicida, con un mayor rendimiento del extracto. Las tasas de inhibición sugieren que L. leucocephala y G. sepium podrían ser evaluadas con la finalidad de reducir la densidad larvaria en pastos.

Reappearance of Mecistocirrus digitatus in cattle from the Mexican tropics: prevalence, molecular, and scanning electron microscopy identification.

Mecistocirrus digitatus is a hematophagous abomasal nematode which causes significant blood and production losses in cattle. The objectives of the present study were to: (1) report the reappearance of M. digitatus in cattle from the Mexican tropics using microscopy, scanning electron microscopy (SEM), and molecular identification, and (2) determine the prevalence of M. digitatus in slaughtered adult cattle from the Mexican tropics. Gastroinestinal nematodes (GIN) were recovered from the abomasum of an 8-yr-old cow (Holstein × Zebu) previously diagnosed with Johne's disease. Of 1,254 GIN, 98.8% were identified as M. digitatus and 1.2% as Haemonchus sp. SEM was used to identify ultrastructure features of M. digitatus (oral cavity, cervical papillae, bursa, bursa lobes papillae, male spicules, anus, and female vulva). A conventional PCR method was used to corroborate the morphological findings. The prevalence of adult cattle infected with M. digitatus and Haemonchus sp., determined from 68 adult cattle from different grazing tropical herds, was 38.2% and 8.8%, respectively. Ninety-eight percent of animals infected with M. digitatus presented lesions in their abomasum such as mucosal inflammation, hemorrhage, and ulcers; some of them had necrosis. The current reappearance of M. digitatus in a Mexican herd suggests the possibility of an underestimated prevalence of this nematode amongst grazing cattle.

In vitro anthelmintic activity of five tropical legumes on the exsheathment and motility of Haemonchus contortus infective larvae.

This study investigated the in vitro anthelmintic (AH) activity of five tropical legume plants [Arachis pintoi CIAT 22160 (A.p. 22160), Gliricidia sepium, Cratylia argentea (C.a. Yacapani), C. argentea CIAT 22386 (C.a. 22386), C. argentea Veranera (C.a. Veranera)] against Haemonchus contortus infective larvae and the role of tannins/polyphenolic compounds in the AH effect. Lyophilized leaf extracts of each plant were evaluated using the Larval Exsheathment Inhibition Assay (LEIA) and the larval migration inhibition assay (LMIA). The role of tannins/polyphenolic compounds in the AH effect was evaluated in both assays using polyethylene glycol (PEG) to remove tannins from the solutions. At the highest concentration (1200µg of extract/ml), A. pintoi 22160, C.a. Yacapani, C.a. Veranera and C.a. 22386 completely inhibited the exsheathment process of H. contortus (P<0.01). At the same concentration (1200µg of extract/ml), the inhibition of larval migration for C.a. 22386, C.a. Veranera and G. sepium was 66.0%, 35.9% and 39.2% (relative to the PBS control), respectively. In both bioassays (LEIA and LMIA), the AH effect shown by each plant was blocked after the addition of polyethylene glycol (PEG), corroborating the role of tannins/polyphenolic compounds.