Caffeoyl and coumaroyl derivatives from Acacia cochliacantha exhibit ovicidal activity against Haemonchus contortus.

ETHNOPHARMACOLOGY RELEVANCE: Acacia cochliacantha is a small tree whose foliage is traditionally used in Mexico for treatment of kidney pain, gastrointestinal illnesses and to kill intestinal parasites. In recent decades, the study of vegetal extracts has offered other possible alternatives for the control of Haemonchus contortus. Considering that this nematode affects dramatically the health and productivity of small ruminants, the aim of this study was to identify the anthelmintic compounds from A. cochliacantha hydro-alcoholic extract (HA-E) through an ovicidal test. MATERIAL AND METHODS: In vitro egg hatch assay was conducted to determinate the anthelmintic effects of a HA-E (60g). Liquid-liquid ethyl acetate/water extraction gave two fractions (EtOAc-F, 1.92g; Aq-F; 58.1g). The less polar compounds from ethyl acetate fraction were extracted by addition of dichloromethane offering a precipitate phase (Mt-F, 1.25g) and a soluble mixture (DCMt-F 1.15g). All fractions were evaluated for ovicidal activity obtaining the egg hatching inhibition (EHI, 0.07-25mg/mL). Ivermectin (0.5mg/mL) was used as a reference drug (positive control), and distilled water, 2.5% DMSO and 2% methanol were used as negative controls. The isolated compounds from the most active fractions were subjected to spectroscopic (1H NMR) Spectrometric (MS) and UV HPLC analysis in order to identify the bioactive compounds. RESULTS: The less polar treatments (AcOEt-F, DCMt-F, DCMt-P) showed the highest ovicidal activities (98-100% EHI; at 0.62-1.56mg/mL) and the major compounds found in these fractions were identified as caffeoyl and coumaroyl derivatives, including caffeic acid (1), p-coumaric acid (2), ferulic acid (3), methyl caffeate (4), methyl-p-coumarate (5), methyl ferulate (6) and quercetin. In case of the less active fractions (Aq-F, Mt-F) were constituted principally by glycosylated flavonoids. CONCLUSION: These results show that caffeoyl and coumaroyl derivatives from Acacia cochliacantha leaves had promising anthelmintic activity against Haemonchus contortus. This leguminous may offer an alternative source for the control of gastrointestinal nematodes of small ruminants.

Cytokine and antioxidant gene profiles from peripheral blood mononuclear cells of Pelibuey lambs after Haemonchus contortus infection.

The expression profiles of cytokines and antioxidant genes were determined from an experimental infection with H. contortus in Pelibuey lambs. The infection was followed for 34 days (d) to determine the number of eggs per gram (epg) and the packed cell volume (PCV). Differential white cell counts and expression profile estimations of IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, FCεR1A, GPX and SOD1 were determined at 0 hour, 4 hours, 2 days and 14 days post-infection (PI) in infected and control groups. Comparison of the fold change between 0 and 4-hours, 4-hours and 2-days and 2- and 14-days periods was performed. Significant differences (P<.05) between epg (>2000) and PCV (>30%) were determined after 21 days and were also observed with regard to monocyte and lymphocyte cells after 2 and 7 days PI. At 0 hour and 14 days PI, the GPX and IL-2 genes showed a 0.37- and 0.49-fold decrease in expression, respectively. In contrast, upregulation was observed at 4 hours of IL-8 (2.58) and FCεR1A (2.71), at 2 days for IL-4 (2.14) and IL-8 (4.02) and at 14 days for IL-2 (0.41), IL-10 (2.35) and FCεR1A (2.28). The comparison between the intervals of infection showed high expression values against H. contortus infection in Pelibuey sheep after the 2nd period of PI involving a dichotomy T cells.

Relative expression analysis of IL-5 and IL-6 genes in tropical sheep breed Pelibuey infected with Haemonchus contortus.

Haemonchus contortus is a parasitic nematode of Pelibuey sheep, a meat breed used in tropical regions. Due to anthelmintic problems, the identification of hosts resistant to H. contortus is another option of control. The aim of this study was to analyse the relative expression of IL-5 and IL-6 genes in Pelibuey sheep after H. contortus infection. Nineteen lambs infected with H. contortus and three more lambs without infection were studied. The haemonchosis was determined by the number of eggs per gram of faeces (epg) and by the estimation of the percentage of the packed cell volume (%pcv). Peripheral blood mononuclear cells (PBMCs) were obtained to extract RNA at 0, 1, 2, 7, 14, 21 and 28 days after infection to quantify the relative expression of IL-5, IL-6 and GAPDH by real-time PCR. Five lambs were classified as low responders (lr) to haemonchosis with averages of 1519 ± 315·3 epg and 31·49 ± 5·13%pcv, and 14 lambs were identified as high responders (hr) with averages of 530 ± 132 epg and 34·88 ± 3·75%pcv. The expression ratio of IL-5 was significantly different compared with control lambs at 2, 7 and 14 days post-infection (PI), and IL-6 was significantly different after 14 days. The highest level of relative expression for IL-5 and IL-6 genes was 9·9-fold and 12-fold after 2 and 14 days for hr hosts (P < 0·05) compared with control group, respectively. In conclusion, the Pelibuey breed in grazing areas exhibited different expression of IL-5 and IL-6 obtained from PBMCs against H. contortus, suggesting the importance of these cytokines in regulating the nematode infection.

Benzimidazole-resistant gastrointestinal nematodes in indigenous Chiapas and Pelibuey sheep breeds from Chiapas, Mexico.

Because of the natural adaptation of Mexican sheep, the aim of the present study was to identify the presence or absence of gastrointestinal parasitic nematodes (GIN) resistant to benzimidazole (BZ) in both Chiapas and Pelibuey sheep breeds on local farms. Both male and female GIN-infected grazing sheep of the two breeds were selected. Sheep faecal samples were collected to obtain infective larvae (L3). This evolving stage of the parasite was used for taxonomic identification of the genus, based on its morphological characteristics. BZ anthelmintic resistance was evaluated using a nematode-compound in vitro interaction bioassay and the allele-specific polymerase chain reaction technique to detect mutations of residues 198 and 200 on isotype 1 of the ß-tubulin gene. Three BZ-based compounds (febendazole (FBZ), tiabendazole (TBZ) and albendazole (ABZ)) at concentrations of 1, 0.5, 0.25, 0.125, 0.062 and 0.03 mg/ml were used to estimate the anthelmintic efficacy and lethal dose (LD50, LD90 and LD99) of the drugs. Two parasitic nematodes, Haemonchus and Teladorsagia, were identified in both isolates. Also, the proportions of anthelmintic resistance identified in GIN of the two sheep breeds were 68% in isolates from the Chiapas breed and 71.8% in the Pelibuey breed. The specific lethal activity obtained with FBZ was higher than 90%. However, TBZ and ABZ showed a lethal activity lower than 50%. High variability in the discriminating dose values was found among the BZ drugs. For example, FBZ LD ranged from 0.01 to 1.20 mg/ml; on the other hand, TBZ and ABZ required a dose ranging from 0.178 to 759 mg/ml. In addition, amino acid changes of Phe (TTC) to Tyr (TAC) at codon 200 of the ß-tubulin gene, showing resistance to BZ, and no changes at codon 198 Glu (GAA) to Ala (GCA) were observed for both isolates. These results confirmed the presence of a genetic mutation associated with BZ in both Chiapas and Pelibuey nematode isolates.