In vitro anthelmintic activity and colocalization analysis of hydroxycinnamic acids obtained from Chamaecrista nictitans against two Haemonchus contortus isolates.

This study assessed the in vitro anthelmintic activity of ethyl acetate extract (Cn-EtOAc) and its bioactive fractions (CnR3 and CnR5) obtained from Chamaecrista nictitans aerial parts against two Haemonchus contortus (Hc) isolates, one resistant (strain HcIVM-R) and another susceptible (strain HcIVM-S) to ivermectin. Ferulic acid and p-coumaric acid were identified in the bioactive fractions; therefore, their commercial standards were also assessed. A colocalization analysis between the ferulic acid commercial standard and eggs of the HcIVM-R strain was performed using confocal laser scanning microscopy and the ImageJ program. The ovicidal effects of the Cn-EtOAc extract, bioactive fractions and commercial compounds were tested through the egg hatching inhibition (EHI) assay on H. contortus isolates HcIVM-R and HcIVM-S. The Cn-EtOAc caused 88 % and 92 % EHI at 5000 µg/mL on HcIVM-R and HcIVM-S, respectively. Fractions CnR3 and CnR5 displayed the highest ovicidal activity against HcIVM-S, with effective concentrations (EC90) of 2134 and 601 µg/mL, respectively. Meanwhile, the commercial standards ferulic acid and p-coumaric acid also resulted in higher effectiveness on the same strain, with EC90 of 57.5 and 51.1 µg/mL. A colocalization analysis of ferulic acid and eggs of HcIVM-R revealed that this compound is localized to the cuticle surface of the embryo inside the egg parasite. The results demonstrated that both ferulic and p-coumaric acids interrupt the egg-hatching processes of the two Hc isolates. Both phenolic acids isolated from C. nictitans and commercial standards exhibited the best anthelmintic effect on HcIVM-S. These findings indicate that the phenolic acids were less effective in egg hatch inhibiting on the HcIVM-R strain compared to the HcIVM-S strain.

Molecular study of the transcription factor SKN-1 and its putative relationship with genes that encode GST and antioxidant enzymes in Haemonchus contortus.

Haemonchus contortus is a parasitic nematode of ruminants. Once inside its host, it is exposed to reactive oxidative species and responds by synthesising antioxidant enzymes as a defence. In Caenorhabditis elegans, antioxidant genes are regulated by the transcription factor skinhead-1 (Cel-SKN-1). However, there is little information about the function of SKN-1 in H. contortus (Hco-SKN-1). Therefore, we performed a molecular investigation to characterise Hco-SKN-1 and its putative relationship with genes encode antioxidant enzymes, namely glutathione S-transferases (Hco-GSTs, n = 3), superoxide dismutase (Hco-SOD) and catalase (Hco-CAT), which are involved in haematophagy and defence against the host. We used in silico sequence analysis of Hco-SKN-1 and Hco-GSTs to design and perform relative expression assays involving H. contortus eggs, infective larvae (L3) and adults. Furthermore, we exposed H. contortus transitional infective larvae (xL3) to erythrocytes or hydrogen peroxide (H2O2) and evaluated the relative expression of antioxidant genes at 24 or 48 h. Gene Ontology (GO) analysis revealed 31 functions associated with Hco-SKN-1 and Hco-GSTs, including stress resistance, larval development and the active immune response. Hco-GST-5957 and Hco-SOD showed the highest expression in adults, indicating a relationship with specific functions at this mature stage. xL3 exposed to erythrocytes or H2O2 showed significant upregulation of Hco-SKN-1, but it occurred after upregulation of the antioxidant genes, indicating that these genes are not regulated by Hco-SKN-1 during the blood-feeding stage. Additional investigation is necessary to understand the putative regulation of antioxidant genes by Hco-SKN-1 during the blood-feeding stage.

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.

Excreted/Secreted 15-kDa Proteins and Serine Protease Peptides from Haemonchus contortus Act as Immune-Response Enhancers in Lambs.

This study assessed the immunoprotective effect in lambs of a native excreted/secreted 15-kDa protein and two synthesised S28 peptides derived from the infective transitory larvae (xL3) and adult stages (AS) of Haemonchus contortus. Twenty-two Pelibuey lambs were divided into negative and positive control groups, as well as immunised lamb groups, with 100 µg of the 15-kDa native protein (15kDaNP) and S28 peptides (S28P). The eggs per gram (EPG) and haematocrit were measured, and AS were counted and morphologically measured. To assess the immunoprotection in lambs, indirect enzyme-linked immunosorbent assays and relative expression analyses of immune cytokines were performed using serum and abomasal samples. Our results showed a 72.28% reduction in adult worms (AW) in the 15kDaNP-immunised group, achieving a high clinical response with 41% haematocrit and low EPG values (436 ± 661). Conversely, the S28P group achieved the highest IgG levels (2.125 ± 0.880 OD), with AW exhibiting the greatest body length (p > 0.05) and upregulation of the IL5 and FCεR1A genes associated with nematode control. The 15kDaNP group showed increased expression of genes related to nematode control and anti-inflammatory responses, including IL4, IL5, IL6, and IL13 (p < 0.05). The S28P and 15kDaNP should be explored as potential vaccines against sheep haemonchosis.

Biochemical parameters, oxidative stress biomarkers, and anatomopathological changes in Wistar rats treated with 3'-demethoxy-6-O-demethylisoguaiacin and norisoguaiacin.

Artemisia cina (Ac) is a plant with anthelmintic compounds such as 3'-demethoxy-6-O-demethylisoguaiacin (D) and norisoguaiacin (N). Three major objectives were proposed: (1) To evaluate biochemical parameters in blood (2) to determine the tissue oxidative stress by biomarkers as TBARS and glutathione peroxidase activity, and (3) to evaluate anatomopathological changes in organs such as the brain, liver, kidney, and lung after oral administration of n-hexane extract of Ac and D and N. D and N were administrated following the OECD guides for acute oral toxicity evaluation (Guide 420). Fifty Wistar rats were distributed into ten groups as follows: Group 1 (G1): 4 mg/Kg; G2: 40 mg/Kg; G3: 240 mg/Kg; G4: 1600 mg/Kg of n-hexane extract of Ac. G5: 2 mg/Kg; G6: 20 mg/Kg; G7: 120 mg/Kg; G8: 800 mg/Kg of D and N, G9: water and G10: polyvinylpyrrolidone at 2000 mg/Kg. At 14 days, the rats were euthanized, and the blood, liver, brain, kidney, and lung were taken for biochemical analysis, anatomopathological changes, and TBARS and GSH evaluation. Glucose, cholesterol, and phosphorus were altered. Histopathological analysis showed multifocal neuronal degeneration in the brain (G2). The kidney and lungs had changes in G7. The GSH and TBARS increased in G6 and G7. The TBARS activity was higher in G1 and G2. In conclusion, extract and D and N of Ac did not have damage at therapeutic doses. D, N, and n-hexane extract of A. cina do not cause histopathological damage at pharmaceutical doses. Still, the brain, kidney, and liver are related to biochemical parameters at higher doses. However, compounds are proposed as antioxidant agents.

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.

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.

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.

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.

In vitro predatory activity of nematophagous fungi isolated from water buffalo feces and from soil in the Mexican southeastern / Atividade predatória in vitro de fungos nematófagos isolados de fezes de búfalos e do solo no sudeste mexicano

Abstract Nematophagous fungi from the feces of water buffalo and soil from southeastern Mexico were isolated, and their in vitro predatory activity against Haemonchus contortus infective larvae (L3) (HcL3) was assessed. The fungi were isolated by sprinkling soil or feces on water agar plates. Six series of 10 Petri dishes containing a 7-day-old culture of each fungus and a series without fungi as the control were prepared. Five hundred HcL3 were added to each plate. The plates were incubated at room temperature. The average of recovered HcL3 was considered to estimate the larval reduction rate. Four nematophagous fungi isolates corresponding to Arthrobotrys oligospora, var microspora (strains 4-276, 269 and 50-80) and one identified as A. oligospora,var. oligospora (isolates 48-80) were obtained from water buffalo feces. From the soil, five isolates were isolated; three corresponded to A. musiformis (Bajío, Yumca and Macuspana isolates), and two isolates were identified as A. oligospora (Comalcalco and Jalapa de Méndez isolates). The predatory activity of isolates from water buffalo feces ranged between 85.9 and 100%. Meanwhile, the fungi from the soil ranged between 55.5 and 100% (p≤0.05). The nematophagous fungi obtained could have important implications in the control of parasites of importance in the livestock industry.

Resumo Fungos nematófagos das fezes de búfalo de água e do solo no sudeste do México foram isolados, e a atividade predatória in vitro contra larvas infectantes de Haemonchus contortus (L3) (HcL3) foi avaliada.Os fungos foram isolados por aspersão de solo e de fezes em placas de agar água. Foram preparadas seis séries de 10 placas de Petri contendo uma cultura de 7 dias de idade de cada fungo e uma série sem fungos como controle. Quinhentos HcL3 foram adicionadas a cada placa. As placas foram incubadas à temperatura ambiente. O número médio de HcL3 recuperadas foi considerado para estimar a taxa de redução larval. Quatro isolados de fungos nematófagos corresponderam a Arthrobotrys oligospora, var microspora (estirpes 4-276, 269 e 50-80) e um isolado identificado como A. oligospora, var. oligospora (isolados 48-80 de fezes de búfalo de água. Do solo, dos cinco isolados três corresponderam a A. musiformis (Bajío, Yumca e Macuspana isolados), e dois isolados foram identificados como A. oligospora (isolados de Comalcalco e Jalapa de Méndez). A atividade predatória de isolados de fezes de búfalo de água variou entre 85,9 e 100%. Enquanto isso, os fungos do solo variaram entre 55,5 e 100% (p≤0,05). Os fungos nematófagos obtidos podem ter importantes implicações nesse controle de parasitos de importância na indústria pecuária.

Cryopreservation of infective larvae of Haemonchus contortus

Se evaluaron tres criopreservadores para mantener larvas infectantes (L3) de Haemonchus contortus bajo condiciones de congelación. Se emplearon tres diferentes soluciones: a) solución salina amortiguadora de fosfatos pH 7.2 (PBS); b) dimetil sulfóxido al 10 por ciento (DMSO); c) glicerina al 10 por ciento. Se colocaron 50,000 L3 por frasco y se introdujeron en nitrógeno liquido, con 10 repeticiones para cada tratamiento. Se determinó la movilidad de L3 a los 30, 90, 180 y 360 días de congelación. Los porcentajes de sobrevivencia fueron: en PBS, 36 por ciento, 20 por ciento, 7 por ciento y 39 por ciento; en DMSO, 87 por ciento, 69 por ciento, 46 por ciento y 85 por ciento; en glicerina, 67 por ciento, 62 por ciento, 29 por ciento y 55 por ciento, en cada tiempo respectivamente. También se detectó la infectividad inoculado larvas criopreservadas a becerros, en DMSO y glicerina, fueron positivos a la Hemoncosis a los 28 días post-contaminación. Con base en el estudio se concluye que el DMSO al 10 por ciento es la solución indicada para criopreservar larvas infectantes de H. contortus