Analysis of P-gp genes relative expression associated to ivermectin resistance in Haemonchus contortus larval stages from in vitro cultures (L3 and xL3) and from gerbils (Meriones unguiculatus) (L4) as models of study.

The aim of the study was to compare the relative gene expression of Haemonchus contortus P-glycoprotein genes (Hco-pgp) between fourth (L4), infective (L3), and transitory infective (xL3) larval stages as laboratory models to study ivermectin (IVM) resistance. The H. contortus resistant to IVM (IVMr) and susceptible to IVM (IVMs) strains were used to develop xL3in vitro culture and to infect Meriones unguiculatus (gerbils) to collect L4 stages. Morphometric differences were evaluated from 25 individuals of H. contortus from each strain. Relative gene expression from xL3 and L4 was determined between comparison of IVMr stages and from IVMr vs IVMs stages. Seven Hco-pgp genes (1, 2, 3, 4, 9, 10, and 16) were analysed by RT-qPCR using L3 stage as control group, per strain, and GAPDH and ß-tubulin as constitutive genes. Morphological changes were confirmed between xL3 and L4 developing oral shape, oesophagus, and intestinal tube. In addition, the body length and width showed statistical differences (p < 0.05). The Hco-pgp1, 2, 3, and 4 genes (p < 0.05) were upregulated from 7.1- to 463.82-fold changes between IVMr stages, and Hco-pgp9 (13.12-fold) and Hco-pgp10 (13.56-fold) genes showed differences between L4 and xL3, respectively. The comparative study between IVMr vs IVMs strains associated to xL3 and L4 displayed significant upregulation for most of the Hco-pgp genes among 4.89-188.71 fold-change. In conclusion, these results suggest the use of H. contortus xL3 and L4 as suitable laboratory models to study IVMr associated with Hco-pgp genes to contribute to the understanding of anthelmintic resistance.

Ruta Chalepensis Full Extract and Organic Phases Exhibit Nematocidal Activity Against Haemonchus Contortus Eggs and Infective Larvae (L3).

The ovicidal and larvicidal effect of a full aqueous extract (FE) and two phases: an aqueous (Aq-Ph) and an ethyl acetate (EtOAc-Ph) from Ruta chalepensis (Rc) stems and leaves against Haemonchus contortus (Hc) were assessed. The egg hatching inhibition (EHI) assay and larval mortality (LM) test were performed by triplicate in 96-well micro-titration plates (n=4 wells). The FE against Hc eggs and larvae was assessed at 1.25, 2.5, 5, 10 and 20 mg/mL; and 30, 60, 90, 120,150 and 200 mg/ mL, respectively. The ovicidal effect of Aq-F and EtOAc-F was assessed at 1.25, 2.5, 5, 10 and 20 mg/mL. Plates were incubated at 28 °C for 48 (ovicidal assay) and 72 h (larvicidal assay). The EHI results were considered based on the mean number of eggs hatching failure after 48 h exposure. The LM was recorded after 72 h exposure to the Rc phases and expressed as mortality percentage. The Rc FE caused 96 and 100% EHI at 10 and 20 mg/mL, respectively; meanwhile, 74% LM was recorded at 200 mg/mL (p<0.05). The Aq-Ph showed 78.5% EHI at 2.5 mg/mL. Likewise, the EtOAc-Ph caused 100% EHI in almost all concentrations. Eighteen compounds including alkaloids, cumarins, triterpens, flavonoids, tannins, saponins and sterols were identified by GC-MS analysis. The results indicate that the aqueous extract from Rc possesses bioactive compounds with in vitro nematocidal activity against Hc; mainly in the EtOAc-Ph. Further studies should be performed to elucidate those compounds searching for alternative methods of control of the sheep haemonchosis.

Acacia farnesiana pods (plant: Fabaceae) possesses anti-parasitic compounds against Haemonchus contortus in female lambs.

Acacia farnesiana pods are rich in secondary metabolites and their biological activities have been recorded as antibacterial, antioxidant and anthelmintic. Previously, an in vitro bioguided study showed the important ovicidal and larvicidal effects of an organic fraction (EtOAc-F) from a hydroalcoholic extract of A. farnesiana pods against Haemonchus contortus. The present study aimed to assess the in vivo anthelmintic effect of EtOAc-F from A. farnesiana pods on the H. contortus faecal egg elimination in female lambs and on the infective larvae (L3) population reduction in coprocultures. The EtOAc-F was obtained from a hydroalcoholic extract from A. farnesiana pods through chromatographic procedures; additionally, some secondary compounds were identified using high-performance liquid chromatography (HPLC). Twenty-one 'Katahdin' crossbred female lambs ranging from three to four months of age, with body weights 21.9 ± 0.39 kg were used. Animals were orally infected with H. contortus (L3) by a single dose of 350 L3/kg BW. Three experimental groups (n = 7) were assigned as follows: 1) Control (untreated), 2) Albendazole, as a positive control (at 7.5 mg/kg BW, unique dose) and 3) EtOAc-F (at 100 mg/kg BW, once every third day, with three applications in total). Individual faecal samples were collected once a week for 5 weeks (at days 38, 45, 52, 59 and 66) post-treatment, to measure the faecal egg counts (FEC) and to obtain the H. contortus (L3) population from faecal cultures. The highest FEC reduction caused by EtOAc-F was 67.7%; meanwhile, albendazole showed a total FEC reduction after the second week post-treatment (day 45). On the other hand, the fraction caused an important reduction in the larval population in coprocultures (54.3-68.5%). The phytochemical analysis revealed the presence of galloyl derivatives and flavonoids as major compounds. The A. farnesiana pods could serve as a natural anthelmintic for the control of H. contortus, and perhaps for controlling other parasites of veterinary importance.

In vitro ovicidal activity of Baccharis conferta Kunth against Haemonchus contortus.

The indiscriminate use of chemical drugs to deworm livestock tends to trigger an anthelmintic resistance problem. In this context, the use of plant extracts rich in secondary metabolites could be an alternative method for the control of gastrointestinal nematodes. Baccharis conferta Kunth is a native plant species from Mexico that is widely used by several ethnic groups as forage for farm animals and medicinally to treat gastrointestinal diseases such as acute stomach ache, dysentery, diarrhoea, vomiting, indigestion, colic, intestinal spasms, urinary problems, and cramps. The aim of the present study was to isolate and characterise the ovicidal constituents of B. conferta and to determine a possible mode of action against Haemonchus contortus. The ovicidal activity was determined using the egg hatching inhibition test (EHI) to assess the methanol extract obtained from B. conferta foliage. The dry extract was partitioned (water/ethyl acetate) to obtain an ethyl acetate (BcEtOAc-F) and aqueous fraction. BcEtOAc-F showed an ovicidal activity of 72.32% EHI at 1 mg/mL. The chromatographic fractionation of BcEtOAc-F resulted in three active sub-fractions with higher ovicidal activity: BcC1R4 (99.15% EHI at 1.0 mg/mL); BcC1R5 (92.51% EHI at 0.75 mg/mL); and BcC1R8 (96.8% EHI at 3.0 mg/mL). Chemical analysis of the BcC1R4 fraction allowed the identification of the major active compound, isokaempferide (1, 98.06% EHI at 1 mg/mL). While, 4,5-di-O-acid caffeoylquinic (3; 96.8% EHI at 3 mg/mL) and an inactive flavone (vicenin-2, 2) were identified as the main compounds in BcC1R8. Chemical characterisation of the isolated compounds was performed via spectroscopic (NMR) and spectrometric (UPLC-MS) analyses. Additionally, the environmental and confocal scanning microscopy analyses revealed that isokaempferide was able to cross the eggshell layer without breaking it and attach itself to the embryo, causing its death. The flavonol, isokaempferide, and the hydroxycinamic acid, 4,5-di-O-caffeoylquinic, displayed powerful ovicidal effects, proving to be a potential alternative for the development of a phytodrug for the control of haemonchosis.

Galloyl derivatives from Caesalpinia coriaria exhibit in vitro ovicidal activity against cattle gastrointestinal parasitic nematodes.

Gastrointestinal nematodes (GIN) are responsible for enormous economic losses worldwide. The use of anthelmintic drugs reduces the parasitic burden in ruminants. However, the excessive use of these drugs triggers anthelmintic resistance in these parasites, which leads to a worrisome inefficacy of most of the commercially available antiparasitic drugs. Caesalpinia coriaria is an arboreal legume possessing medical properties, although the antiparasitic potential of this plant against animal parasitic nematodes has not yet been studied. The aim of this study was to assess the in vitro ovicidal activity of a hydro-alcoholic extract (HA-E) from C. coriaria fruits against GIN and to identify the compounds responsible for this activity through an egg hatch inhibition (EHI) assay. GIN eggs obtained from cattle faeces were used in bio-guided assays. The HA-E was subjected to a liquid-liquid extraction using water and ethyl acetate to obtain two fractions, an organic fraction (EtOAc-F, 27% yield) and an aqueous (Aq-F, 73% yield) fraction. The chromatographic fractionation of the EtOAc-F (2 gr) was performed on a glass column packed with silica gel and eluted with dichloromethane/methanol with 10% ascending polarity. The bioactive compounds were analysed using high-performance liquid chromatography (HPLC) with UV detection, nuclear magnetic resonance (NMR) spectroscopy and mass spectroscopy (MS). The HA-E extract and the EtOAc-F showed ovicidal activity at a LC50 of 0.92 and 0.16 mg/mL, respectively. A concentration-dependant effect was observed in both treatments. Chromatographic fractionation of the EtOAc-F, allowed for the isolation and characterisation of three important compounds: methyl gallate (1), gallic acid (2) and an unidentified compound (UC). The bioactive molecules (2 and UC) displayed an ovicidal activity close to 100% at 1 mg/mL concentration. The results of this work show that gallic acid (2) isolated from C. coriaria fruits is responsible for its ovicidal activity. The use of Caesalpinia coriaria could be explored in future studies as an environmentally-friendly alternative for the control of GIN in ruminants.

Effects of different extracts of three Annona species on egg-hatching processes of Haemonchus contortus.

This study assessed the in vitro anthelmintic (AH) activity of methanol and acetone:water leaf extracts from Annona squamosa, A. muricata and A. reticulata against Haemonchus contortus eggs. The egg hatch test was used to determine the effective concentrations required to inhibit 50% of eggs hatching (EC50). The role of polyphenols on AH activity was measured through bioassays with and without polyvinylpolypyrrolidone (PVPP). Methanolic extracts mainly caused the death of eggs at the morula stage (ovicidal activity). Meanwhile, acetone:water extracts caused egg-hatching failure of developed larvae (larvae failing eclosion (LFE) activity). The lowest EC50 values against H. contortus eggs were observed for the methanolic extracts from A. reticulata and A. muricata (274.2 and 382.9 µg/ml, respectively). From the six extracts evaluated, the methanolic extracts of A. muricata, A. reticulata and A. squamosa showed the highest ovicidal activity, resulting in 98.9%, 92.8% and 95.1% egg mortality, respectively. When the methanolic extract of A. squamosa was incubated with PVPP, its AH activity increased. Similarly, when acetone:water extracts of A. muriata and A. reticulata were incubated with PVPP, their LFE activity increased. Alkaloids were only evident in methanolic extracts, irrespective of PVPP incubation. The presence of acetogenins was not observed. In conclusion, methanolic extracts obtained from leaves of A. muricata, A. reticulata and A. squamosa showed ovicidal activity affecting the morula of H. contortus eggs, with minor LFE activity. Meanwhile, acetone:water extracts showed mostly LFE activity, with a lower proportion of ovicidal activity.

In vitro nematicidal effect of Chenopodium ambrosioides and Castela tortuosa n-hexane extracts against Haemonchus contortus (Nematoda) and their anthelmintic effect in gerbils.

The in vitro nematicidal effect of Chenopodium ambrosioides and Castela tortuosa n-hexane extracts (E-Cham and E-Cato, respectively) on Haemonchus contortus infective larvae (L3) and the anthelmintic effect of these extracts against the pre-adult stage of the parasite in gerbils were evaluated using both individual and combined extracts. The in vitro confrontation between larvae and extracts was performed in 24-well micro-titration plates. The results were considered 24 and 72 h post confrontation. The in vivo nematicidal effect was examined using gerbils as a study model. The extracts from the two assessed plants were obtained through maceration using n-hexane as an organic agent. Gerbils artificially infected with H. contortus L3 were treated intraperitoneally with the corresponding extract either individually or in combination. The results showed that the highest individual lethal in vitro effect (96.3%) was obtained with the E-Cham extract at 72 h post confrontation at 40 mg/ml, followed by E-Cato (78.9%) at 20 mg/ml after 72 h. The highest combined effect (98.7%) was obtained after 72 h at 40 mg/ml. The in vivo assay showed that the individual administration of the E-Cato and E-Cham extracts reduced the parasitic burden in gerbils by 27.1% and 45.8%, respectively. Furthermore, the anthelmintic efficacy increased to 57.3% when both extracts were administered in combination. The results of the present study show an important combined nematicidal effect of the two plant extracts assessed against L3 in gerbils.

Influence of the physiological stage of Blackbelly sheep on immunological behaviour against gastrointestinal nematodes.

The influence of the physiological stage of a Blackbelly flock against natural infection by gastrointestinal parasitic nematodes (GIN) was investigated. Data on serum and saliva IgA levels and peripheral cellular immune response were recorded in 51 sheep for 6 months. The flock was divided based on their physiological stage as follows: a) pregnant ewes, b) lactating ewes, c) non-pregnant ewes, d) pre-weaning lambs, e) growing lambs and f) replacement sheep. Additionally, sheep were classified based on the parasite infection as stabled or grazing management system as follows: g) non-infected stabled sheep, h) infected and stabled sheep and i) infected grazing sheep. Faeces and blood samples were collected every fifteen days. The percentage of packed cell volume (PCV), plasma protein, (PP) and leucocyte differential counts (LDC) were recorded. Likewise, serum and saliva IgA levels were measured by the indirect ELISA technique. The number of GIN eggs excreted per g of faeces (EPG) was also determined using the McMaster technique. Data were analysed using the SAS program to identify fixed effect of physiological stages, infection time and interactions. Results showed high EPG values in stabled lactating ewes (2764) and in growing lambs (2950-4550 EPG). Pregnant ewes had higher PCV values (27%) than lactating ewes (23.5%). Infected lambs showed low PCV values (28.5%) and high PP values (5.9 g/dL) compared with non-infected lambs (31% PCV and 5.6 g/dL PP). The two most abundant GIN species identified were Haemonchus contortus (91%) and Trichostrongylus colubriformis (81%). Pregnant and non-pregnant ewes showed 29% and <20% of IgA values, respectively. High EPG, low IgA and eosinophils counts were observed until 180 days of age in infected lambs. In conclusion, the group of infected lactating ewes and lambs showed susceptibility to GIN than the other groups, including the pregnant ewes.

In vitro larvicidal and in vivo anthelmintic effects of Oxalis tetraphylla (Oxalidaceae) hydroalcoholic extract against Haemonchus contortus in lambs.

The in vitro larvicidal and in vivo anthelmintic effects of Oxalis tetraphylla hydroalcoholic extract (HE), against Haemonchus contortus in experimentally infected lambs, were assessed. We used a microtitration plate method, comprising the following two stages. Stage 1: 20 µl of water containing 200 sheathed H. contortus infective larvae (ShHcl) were deposited in every well of three series; then, the series 2 and 3 wells were treated with 80 µl 1% ivermectin and O. tetraphylla HE at 20 mg/ml, respectively. Stage 2: the same procedure was performed replacing the ShHcl with exsheathed larvae (ExShHcl). Evaluations were performed after 24 and 48 h. The total numbers of dead and live larvae were counted. A second experiment evaluated the reduction in nematode egg populations in the faeces of lambs treated orally with the O. tetraphylla HE. The 27 lambs used were divided into Groups 1, 2 and 3 (n = 9), which were administered water (positive control), levamisole 1 m (7.5 mg/kg body weight (BW), as a unique dose) and O. tetraphylla HE (20 mg/kg BW), respectively. The plant HE was administered daily for 8 days. The in vitro assay showed 80.9% and 86.5% larval mortality of ShHcl after 24 and 48 h, respectively, while the corresponding mortality values for ExShHcl were 97 and 99%, respectively. The in vivo assay showed variability in the eggs/gram of faeces (epg) values; however, at the end of the trial, the average reduction in the epg values of the O. tetraphylla HE group was 45.6% (P < 0.05). Oxalis tetraphylla HE contains compounds that belong to the flavonol group with anthelmintic activity.

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.

Consumption of nutritional pellets with Duddingtonia flagrans fungal chlamydospores reduces infective nematode larvae of Haemonchus contortus in faeces of Saint Croix lambs.

Two groups of six Haemonchus contortus infected Saint Croix lambs each received different diets for 11 weeks: control group, commercial food, molasses and lucerne hay; and treated group, nutritional pellets (NPs) containing Duddingtonia flagrans at 2 × 106 chlamydospores/kg body weight (BW), sorghum and lucerne hay. Mean BW gain (BWG), body condition score (BCS) and packed cell volume (PCV) and also eggs/g of faeces (EPG) and recovered L3 were compared using a repeated measures across time model. Groups had similar BWG (control 139.7 ± 0.035 g/day and treated 167.7 ± 0.041 g/day), BCS (control 3.6 ± 0.39 and treated 3.4 ± 0.46) and PCV (control 32.5 ± 1.68% and treated 30.0 ± 1.68%). The mean EPG of the control group was 1215 ± 1040 and in the treated group it was 2097.91 ± 2050. No reduction in larval population was observed during weeks 2 and 3. The greatest larval population reduction in the faeces of treated lambs was observed during the first week (70.5%) and from weeks 6 to 11, with a mean value close to 70% (P < 0.05). In general, both experimental groups showed a similar feed conversion. It was concluded that both diets resulted in similar lamb growth, PCV, BCS and H. contortus EPG. However, NP consumption significantly reduced the H. contortus L3 population in lamb faeces.

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.

Duddingtonia flagrans chlamydospores in nutritional pellets: effect of storage time and conditions on the trapping ability against Haemonchus contortus larvae.

The study evaluated the effect of storage time and conditions of nutritional pellets (NP) containing Duddingtonia flagrans chlamydospores on its in vitro trapping ability against Haemonchus contortus L3 larvae. The treated batch (200 NP) contained 4 × 106 chlamydospores of the FTH0-8 strain, whereas the control batch (200 NP) was produced without spores. Both NP batches were exposed to four experimental storage conditions: (T1) shelves (indoors); (T2) refrigeration (4°C); (T3) outdoors under a roof; and (T4) 100% outdoors. Each group comprised 48 NP with spores and 48 NP without spores (control). The ability of D. flagrans spores to trap H. contortus L3 larvae was evaluated for 8 weeks for each storage condition. For that purpose, six randomly selected NP with spores were compared to their respective control NP. Each NP was individually crushed. The crushed material (1 g) was placed on the surface of a 2% water agar plate with 200 H. contortus L3 larvae. Plates were sealed and were incubated at room temperature for 8 days. The whole content of every plate was transferred to a Baermann apparatus to recover the remaining larvae. There was a clear larval reduction in the NP with spores, compared to the respective control NP in the four storage conditions (P< 0.05). The mean reductions ( ± SEM) of the storage conditions were 67 ± 4.9 (T2), 77 ± 6.1 (T1), 81.5 ± 3.8 (T4) and 82.1 ± 2.5 (T3). Larval reductions were similar at all times and were not affected by storage conditions or storage time (R 20.05). The long-term shelf-life of the chlamydospores in the NP suggests that this spore dosage technology is a viable option.

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.

Evaluation of predation of the mite Lasioseius penicilliger (Aracnida: Mesostigmata) on Haemonchus contortus and bacteria-feeding nematodes.

Predation by the mite Lasioseius penicilliger was studied on three nematode species, i.e. infective larval stages (L3) of Haemonchus contortus and adults of Panagrellus redivivus and Rhabditis sp. Experiments were carried out in 5.5-cm diameter Petri dishes containing 2% water-agar over a period of 5 days. Batches of up to 1500 third-stage larvae (L3) of H. contortus and 1000 adult nematodes of P. redivivus and Rhabditis sp. were exposed to five mites in separate Petri dishes. Upon contact, each mite used its pedipalp and legs to identify and hold its prey and then used its chelicerae to feed upon the prey. Predation by L. penicilliger was chance dependent but mites became aggregated around any injured/damaged prey, thereby suggesting some form of chemoperception. The rate of predation on the three species of nematodes was high but L3 of H. contortus and adult Rhabditis sp. were preferred.

Phytochemical profile and nematicidal activity of a hydroalcoholic extract from Cazahuate flowers (Ipomoea pauciflora M. Martens & Galeotti) against Haemonchus contortus infective larvae.

Haemonchus contortus (Hc) is a hematophagous parasite affecting the health and productivity of flocks. The administration of chemical anthelmintic drugs (AH) is the common method of deworming; however, generates resistance in the parasites to AH and it is a public health risk due to drug residues in milk, meat and sub-products. Natural compounds from plants are explored to diminish this parasitosis, improving their health and productivity, without the negative effects of AH. Ipomoea genus is a group of climbing plants belonging to the Convulvulaceae family possessing perennial leaves and tuberous roots. Medicinal properties has been attributed to this plant including nutritional agents, emetics, diuretics, diaphoretics, purgatives and pesticides. The objective of this study was assessing the in vitro nematocidal activity of a hydroalcoholic extract (HA-E) obtained from Ipomoea pauciflora (Cazahuate) flowers against Hc infective larvae (L3) and to identify its phytochemical profile (PhC-P). The assay was carried out using microtiter plates (MTP). Four HA-E concentrations were assessed and Ivermectin and distilled water were used as positive and negative control groups, respectively. Approximately 100 Hc L3 were deposited in each well (n=12) and incubated at 25-35°C for 7 days. Data were analyzed using ANOVA and a General Linear Model (GLM) followed by Tukey test (P<0.05). The treatments showing a concentration-dependent effect (CDE) were analyzed to identify their 50% and 90% lethal concentrations (CL50, 90) via a Probit Analysis. The highest mortality was observed at 50 mg/mL (82.64 ± 0.71%) and the lowest at 6.25 mg/mL (56.46 ± 2.49%), showing a CDE with increasing mortality from 6.25 to 50 mg/mL. The PhC-P revealed the presence of alkaloids, coumarins, flavonoids, tannins and triterpenes/ sterols. A HA-E from flowers of I. pauciflora will be considered to assess its potential use in the control of haemonchosis in small ruminants.

Anthelmintic effects of Prosopis laevigata n-hexanic extract against Haemonchus contortus in artificially infected gerbils ( Meriones unguiculatus).

The anthelmintic effect of Prosopis laevigata (mezquite) n-hexanic extract was evaluated against Haemonchus contortus endoparasitic stages in artificially infected gerbils (Meriones unguiculatus). Prosopis laevigata leaves were collected from the Sierra de Huautla, Ecological Reserve of the Biosphere, in Morelos State, Mexico; dehydrated under shade and macerated with n-hexane for 3 days, followed by distillation for 8 h. This procedure was repeated three times and the final extract was kept at 4 degrees C. The in vivo effect of the plant extract was evaluated in gerbils artificially infected with H. contortus. Plant extract concentration was 40 mg/ml. Three groups of gerbils were as follows: group 1 (n = 7), P. laevigata extract at 100 microl intraperitoneally (IP); group 2 (n = 6), control--Tween 20 in water at a single dose of 100 microl IP; group 3 (n = 8) also served as a control, receiving water only, to determine the mortality due to causes other than the plant extract. An additional group of seven gerbils (group 4) was administered fenbendazole, as a positive control. Five days later the animals were euthanized and stomach and mucosa removed to quantify the nematodes. Data were analysed using the Student's t-test to compare the mean of nematodes obtained in groups 1, 2 and 3. The parasite population in the plant extract treated group 1 was reduced by 42.5% (P < 0.05) with respect to the control group 2; and when control group 3 was used for comparison the parasitic reduction was estimated as 53.11%. This study shows the in vivo anthelmintic effect of P. laevigata n-hexane extract for the first time, using gerbils as an in vivo model, with potential use in sheep.

Galloyl flavonoids from Acacia farnesiana pods possess potent anthelmintic activity against Haemonchus contortus eggs and infective larvae.

ETHNOPHARMACOLOGY RELEVANCE: Acacia farnesiana (L.) Willd is a shrub legume used as condiment, medicinal plant and bioactive herbage. This species is used in traditional medicine of several countries to relieve the symptoms of gastrointestinal diseases, diarrhoea, stomach pain and typhoid as well as astringent, antidysenteric and anthelmintic. Some studies have shown that this plant displayed anthelmintic activity against several gastrointestinal nematode parasites of livestock, and also against parasites of human beings, such as malaria. AIM OF THE STUDY: This work describes the isolation and chemical identification of the anthelmintic compounds of Acacia farnesiana pods against eggs and infective larvae of the sheep parasitic nematode Haemonchus contortus. The bio-guided chemical fractioning of A. farnesiana pods using ethyl acetate against H. contortus eggs and infective larvae allowed for the identification of naringenin 7-O-(6″-galloylglucoside) (flavonol group) as the compound responsible for the anthelmintic activity against this important parasitic nematode. MATERIALS AND METHODS: Anthelmintic activity was assessed using the egg hatching inhibition assay (EHI) and mortality tests. A complete hydroalcoholic extract (HA-E) at 12.5-50 mg/mL, an aqueous fraction (Aq-F) at 3.12-25 mg/mL and an ethyl acetate fraction (EtOAc-F) at 3.12-25 mg/mL were analysed in the first selection phase. The purification of compounds through the chromatographic separation of the organic fraction resulted in nine less complex mixtures (C1F1, C1F2, C1F3, C1F4, C2F1, C2F2, C2F3, C2F4 and C2F5) that were assessed at 0.62-5 mg/mL concentrations. In addition, thiabendazole (0.6 mg/mL) and ivermectin (5 mg/mL) were used as positive controls. Likewise, distilled water and 4% methanol were used as negative controls. The bioactive compounds of EtOAc-F were obtained and characterised through chromatographic processes like open column chromatography, thin layer chromatography (TLC), high performance liquid chromatography (HPLC), ultra-performance liquid chromatography (UPLC) and gass chromatography-mass detection (GC-MS). Bioactive compounds were identified by spectroscopy (1H and 13C NMR) and mass spectrometric analysis. Additionally, the H. contortus eggs and infective larvae exposed to the bioactive compounds were observed through environmental scanning electron microscopy (ESEM) and confocal laser scanning microscopy (CLSM). Data were analysed based on a completely randomised design using ANOVA through a general linear model. RESULTS: The EtOAc-F fraction showed the highest ovicidal and larvicidal activities, at close to 100% at 3.12 and 6.25 mg/mL, respectively. The treatments C1F2, C1F3 and C2F3 displayed the main ovicidal activity (80-100%) at 2.5 mg/mL. The major compounds found in these sub-fractions were identified as galloyl derivatives and flavanones, including gallic acid (1), methyl gallate (2), ethyl gallate (3), naringin (4), naringenin 7-O-(4″, 6″-digalloylglucoside) (5), naringenin 7-O-(6″-galloylglucoside) (6) and naringenin (7). Likewise, the ESEM and CLSM images showed that the assessed compounds adhered to the eggshell and the external cuticle of the larvae. CONCLUSION: These results indicate that A. farnesiana pods contain nematocidal compounds and might be promising natural anthelmintic agents against H. contortus. This leguminous plant could be used as a nutraceutical food source for the control of gastrointestinal nematodes in small ruminants.

Anthelmintic-like activity of polyphenolic compounds and their interactions against the cattle nematode Cooperia punctata.

Polyphenolic compounds (PCs) have been proposed as one of the most bioactive group of secondary metabolites occurring in nature and have been associated to anthelmintic (AH)-like activity of plants against cattle nematodes. However, little is known regarding their synergetic / antagonistic interactions. This study assessed the in vitro AH-like activity of commercial PCs: quercetin, caffeic acid, rutin and coumarin, and their combinations against the egg hatching and larval exsheathment of Cooperia punctata; one of the most prevalent nematodes affecting grazing cattle in tropical regions. The molecules selected for the in vitro analysis were identified as bioactive phytochemicals of plants through bio-guided fractionation in previous studies. To estimate mean effective concentrations (EC50) five increasing concentrations were used for both Egg hatching inhibition assay (EHIA) and larval exsheathment inhibition assay (LEIA) (0.6-9.8 mg mL-1 and 0.15-2.4 mg mL-1, respectively). From the four molecules, only rutin did not affect egg hatching; while quercetin, showed no bioactivity against eggs or larvae (P > 0.766 and P > 0.621, respectively). Best-fit EC50 estimated through the EHIA was considered for PCs classification as bioactive (coumarin and caffeic acid) and non-bioactive (quercetin and rutin). Phytochemical interactions were subsequently assessed combining bioactive:non-bioactive PCs (8:2 ratio), and the nature of their interaction was classified using the fractional inhibitory concentration index (FICindex). Combinations had a highly synergistic interaction against larval exsheathment (FICindex < 0.5) except for coumarin:rutin against egg hatching (FICindex> 0.5). Quercetin and rutin acted as PCs AH-like activity enhancers, reducing EC50 of bioactive molecules in a range of 43%-64% and 68%-83% for EHIA and LEIA, respectively. A linear relationship between low molecular weight of molecules and ovicidal activity was observed; where, molecules with lower molecular weight displayed better-fit EC50 for ovicidal activity. Furthermore, coumarin and caffeic acid bioactivity against free-living stages of C. punctata makes them suitable candidates as markers for anthelmintic-like activity in bioactive forages. Combinations used through this investigation showed a potent anthelmintic-like activity against free-living forms of C. punctata, representing a first step towards the identification of promising alternatives for nematode control.

A technique for the quantification of Duddingtonia flagrans chlamydospores in sheep faeces.

Previous observations showed that Duddingtonia flagrans chlamydospores were visualized in McMaster chambers containing faeces of treated sheep. This trial explored the McMaster technique as a tool to quantify chlamydospores in sheep faeces. A range of individual chlamydospore doses (from 19.5 x 10(6) to 177.5 x 10(6)) were offered orally to nine lambs for 7 consecutive days. A faecal sample (5 g) was daily obtained from the rectum of each animal (from days 1 to 13) to perform the McMaster technique using a sugar flotation fluid with 1.27 g/mL density. Each chlamydospore counted in the McMaster chamber was considered as 50 chlamydospores per g of faeces (CPG). The results confirmed that the estimated CPG was associated with the daily dose offered to the animals (r(2)=0.90; P<0.001). Furthermore, the total chlamydospore dose received by each animal was strongly associated to the total quantity of CPG obtained from the bulk faeces (TCtot) (r(2)=0.96; P<0.0001). Quantification of CPG can be used as a helpful tool to determine the number of chlamydospores reaching the faeces in orally dosed animals. This could be used to evaluate the efficacy of D. flagrans for the control of gastrointestinal nematode larvae in sheep faeces.