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.

Effect of an Arthrobotrys musiformis (Fungi: Orbiliales) culture filtrate on the population of gastrointestinal parasitic nematode eggs in faeces of grazing lambs.

This study assessed the anthelmintic activity of the oral administration of a free-spore culture filtrate of the nematophagous fungus (NF) Arthrobotrys musiformis (M-10) on gastrointestinal parasitic nematodes (GIN) in naturally infected lambs. The fungus was grown on potato-dextrose agar plates (PDA) and transferred to a fermented rice medium (FRM). After 40-day incubation the total amount of FRM with the growing fungi was transferred to a flask shaker with distilled water for a 24 h period. The fungus was centrifuged and filtered. Three groups of six naturally-infected lambs (>1000 epg) each were treated once as follows: Group 1) 63.8 mg/kg A. musiformis culture filtrate (CF) (per os); Group 2) Levamisole 7.5 mg/ml (intramuscularly), Group 3) 15 ml of distilled water (per os). Faecal samples were individually collected on days -2, 0, 2, 4, 6, 8 and 10 after treatment. For each experimental group, mean egg shedding was calculated and transformed (log 10 [epg + 1]). Means between the fungal filtrate group and the negative control were analysed using a T-Student Test. The faecal egg count reduction test (FECRT) was performed in groups treated with CF and Levamisole in relation to the control group (water) were 36.8-57.4% and 89-95.4%, respectively., although due to the difference between groups, no statistical significance was found (p > 0.05). The use of A. musiformis CF appears to be a good alternative treatment, although, more studies should be performed to establish the use of these fungal products as potential tools for GIN control.

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.

Molecular marker sequences of cattle Cooperia species identify Cooperia spatulata as a morphotype of Cooperia punctata.

The genus Cooperia includes important parasites of ruminants and currently contains 34 accepted species. However, even for those species infecting livestock, there is a considerable lack of molecular information and many species are only identifiable using subtle morphological traits. The present study aimed to provide molecular data to allow diagnosis of Cooperia species infecting cattle. Partial sequences of two mitochondrial (cytochrome oxidase 2, 12S rRNA gene) and two nuclear genes (isotype 1 ß tubulin gene including two introns, internal transcribed spacers (ITS) were obtained from morphologically identified specimens of Cooperia pectinata, Cooperia punctata and Cooperia spatulata as well as from larvae of pure Cooperia oncophora and C. punctata laboratory isolates. Pairwise identity of ITS-2 sequences was very high and it was the only region able to identify a specimen as Cooperia sp. However, the ITS-2 was unreliable for diagnosis at the species level. All other marker sequences could not unequivocally be allocated to the genus Cooperia but allowed clear species identification with the exception of the pair C. punctata/C. spatulata for which no significant differences were found for any marker sequence. Maximum-likelihood phylogenetic analyses of individual genes as well as a multi-locus analysis covering all four sequences confirmed that specimen identified as C. spatulata were randomly distributed throughout the C. punctata cluster and formed no group of their own. In contrast, the other Cooperia species formed clearly separated and statistically supported clusters. These data indicate that C. spatulata is most likely only a morphotype of C. punctata and the name should be considered a synonym. Combinations of nuclear and mitochondrial markers should be used to identify morphotypes or cryptic species to benefit from excellent barcoding properties of the latter but allowing proper phylogenetic analyses and controlling for lineage sorting that might occur for mitochondrial genotypes within a species.

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.

Anthelmintic effect of 2H-chromen-2-one isolated from Gliricidia sepium against Cooperia punctata.

Gliricidia sepium is a tropical legume with known anthelmintic-like properties. The aim of this study was to: (1) perform a bio-guided fractionation of an acetonic extract of G. sepium leaves using the egg hatch assay (EHA); (2) elucidate the anthelmintic (AH)-like phytochemical using nuclear magnetic resonance (NMR); and (3) assess the ultrastructural damage of the Cooperia punctata treated eggs. The anthelmintic activity of G. sepium was traced from an acetonic extract using the EHA. Phytochemicals were isolated through silica gel columns and elucidated through spectroscopic measurements (1H and 13C). Final fraction was evaluated with EHA at decreasing concentrations of: 1.100; 0.500, 0.250, 0.125, 0.060, 0.001 and 0.00001 mg mL-1. Egg hatching inhibition was calculated using the formula: 100*(1-HT/HC). The maximal half of effective concentration (EC50) was calculated with GraphPad. Bio-guided isolation procedures lead to the elucidation of 2H-chromen-2-one, which inhibited both hatching and embryo development of C. punctata (EC50 of 0.024 ± 0.082 mg mL-1) (P < 0.05). Scanning and Transmission Electron Microscopy (SEM and TEM) revealed electrodensity alterations and fractures in the eggshell layers. After toxicity evaluations and in vivo assessment, 2H-chromen-2-one can be suggested as a novel AH-phytochemical for reducing larval density in pastures and worm burdens inside the host.

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.

Elucidation of Leucaena leucocephala anthelmintic-like phytochemicals and the ultrastructural damage generated to eggs of Cooperia spp.

Leucaena leucocephala is a tropical forage legume suggested as an alternative method to control gastrointestinal parasitism in ruminants. This study: (1) performed a bio-guided fractionation of an aqueous extract of L. leucocephala using the egg hatch assay (EHA) to identify the anthelmintic (AH)-like phytochemicals present in fresh leaves, and (2) assessed the ultrastructural damage to eggs of Cooperia spp. after incubation with the final fraction. Phytochemicals were isolated using silica gel columns and identified using high performance liquid chromatography and standards for comparison. The final fraction was evaluated using EHA at 0.06, 0.125, 0.250, 0.500 and 1.1 mg ml(-1). The lethal concentration to inhibit 50% of Cooperia spp. egg hatching (LC50) was calculated using a Probit analysis. Scanning and transmission electron microscopy revealed the ultrastructural changes present in Cooperia spp. eggs. Bio-guided isolation procedures led to the recognition of an active fraction (LlC1F3) mainly composed of quercetin (82.21%) and caffeic acid (13.42%) which inhibited 90.49 ± 2.8% of Cooperia spp. egg hatching (P<0.05), and an LC50 of 0.06 ± 0.14 mg ml(-1). Scanning electron microscopy (SEM) showed eggs exposed to the active fraction had an irregular external layer with small projections and ruptures of lateral eggshell walls. Transmission electron microscopy (TEM) showed changes to Cooperia spp. eggs in electro-density, including the thickness of the eggshell layers and fractures after incubation with the final fraction (LlC1F3). Changes in bioactivity after purification suggest synergistic interactions between quercetin and caffeic acid.

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.