Haemonchus Contortus Infective Larvae Research Articles

In vitro ovicidal and larvicidal activity of a hydroalcoholic extract and its fractions from Cyrtocarpa procera fruits on Haemonchus contortus

This study describes the in vitro anthelmintic effect of a hydroalcoholic extract (HA-E) and its fractions from Cyrtocarpa procera fruits against Haemonchus contortus eggs and infective larvae. The HA-E was subjected to bipartition using ethyl acetate, which resulted in an aqueous fraction (Aq-F) and an organic fraction (EtOAc-F). The HA-E and both fractions were tested using the egg hatching inhibition assay (EHIA) and the larval mortality test (LMT). Fractionation of the EtOAc-F was achieved using different chromatographic processes, i.e., open glass column and HPLC analysis. Fractionation of the EtOAc-F gave 18 subfractions (C1R1-C1R18), and those that showed the highest yields (C1R15, C1R16, C1R17 and C1R18) were subjected to anthelmintic assays. The HA-E and the EtOAc-F displayed 100% egg hatching inhibition at 3 and 1 mg/mL, respectively, whereas Aq-F exhibited 92.57% EHI at 3 mg/mL. All subfractions tested showed ovicidal effect. Regarding the larval mortality test, HA-E and EtOAc-F exhibited a larvicidal effect higher than 50% at 50 and 30 mg/mL, respectively. The subfractions that showed the highest larval mortality against H. contortus were C1R15 and C1R17, with larval mortalities of 53.57% and 60.23% at 10 mg/mL, respectively. Chemical analysis of these bioactive subfractions (C1R15 and C1R17) revealed the presence of gallic acid, protocatechuic acid, and ellagic acid. This study shows evidence about the ovicidal and larvicidal properties of C. procera fruits that could make these plant products to be considered as a natural potential anthelmintic agents for controlling haemonchosis in goats and sheep.

The in vitro rumen exsheathment test for studying the effect of plant extracts on the exsheathment of Haemonchus contortus infective larvae

This study applied the in vitro rumen exsheathment test (IVRET) to evaluate the exsheathment kinetics of Haemonchus contortus infective larvae (L3) incubated in ruminal liquor (RL) containing acetone:water extracts of Acacia pennatula (AP), Gymnopodium floribundum (GF), Havardia albicans (HA) or Lysiloma latisiliquum (LL). The role of polyphenols in the biological activity of the evaluated extracts was also determined. Larvae were incubated in RL either alone or added with a different plant extract (AP, GF, HA, or LL) at 1200 μg/mL. Polyethylene glycol (PEG) was added to block polyphenols in each treatment (RL+PEG, AP+PEG, GF+PEG, HA+PEG, and LL+PEG). After incubation times of 0, 1, 3, 6, 9, and 24 h, the exsheathment process was stopped to count the number of ensheathed and exsheathed L3. A Log-Logistic model was used to determine the L3 exsheathment kinetics in the different RL treatments. The inflection point of the respective kinetic curves, which indicates the time to reach 50 % exsheathed L3 (T50), was the only parameter that differed when comparing the exsheathment models (99 % probability of difference). The T50 values obtained for GF, HA, and LL treatments (T50 = 7.11 – 7.58 h) were higher in comparison to the T50 of RL (5.72 h) (≥ 70 % probability of difference). The L3 incubated in RL added with GF, HA, and LL extracts delayed their exsheathment at 3 and 6 h of incubation (28.71 – 48.06 % exsheathment reduction) compared to the RL treatment. The T50 value for AP, AP+PEG, GF+PEG, HA+PEG, and LL+PEG were similar to RL and RL+PEG (T50 = 5.34 – 6.97 h). In conclusion, the IVRET can be used to identify plants with the potential to delay the exsheathment of H. contortus L3 in the ruminal liquor. The acetone:water extracts of G. floribundum, H. albicans, and L. latisiliquum delayed the T50 of H. contortus exsheathment, which was evident at 3 and 6 h of incubation in ruminal liquor. The observed exsheathment delay was attributed to the polyphenol content of the extracts.

Adapting the in vitro rumen incubation method to evaluate the effect of a plant extract on the exsheathment inhibition of Haemonchus contortus infective larvae

This study adapted the in vitro rumen incubation (IVRI) method to evaluate the biological activity of a Gymnopodium floribundum leaves extract against the exsheathment of Haemonchus contortus infective larvae (L3), and to determine the role of plant polyphenols on the biological activity. The incubation protocol followed the IVRI method, adding polyethylene glycol (PEG) as a polyphenol-blocking agent. The L3 were incubated in ruminal liquor (RL), ruminal liquor with PEG (RL+PEG), ruminal liquor with G. floribundum extract (RLE), and ruminal liquor with G. floribundum extract and PEG (RLE+PEG). Incubation condition controls included phosphate buffered saline (PBS), PBS with PEG (PBS+PEG), incubation medium (without ruminal liquor) (IM), and incubation medium with PEG (IM+PEG). The L3 were recovered after incubation times of 0, 1, 3, 6, 9, and 24 h (39 °C). The respective L3 exsheathment kinetics were estimated for the different treatments (RL, RL+PEG, RLE, and RLE+PEG) using Log-Logistic models. The parameters of the different models were compared to determine the impact of the extract, with or without PEG, on the L3 exsheathment kinetics. The exsheathment in PBS and PBS+PEG remained < 2.71% at each incubation time. The exsheathment in IM and IM+PEG reached 13.58% and 17.18% at 24 h, respectively. The exsheathment percentages for RLE were lower than those for RL at 3, 6 and 9 h of incubation. The inflection point, indicating the time required to reach 50% of the maximal exsheathment (T50), was the only parameter that differed between the ruminal liquor models. The T50 in RLE (7.106 h) was higher than the values obtained for RL (5.385 h) and RL+PEG (4.923 h) (99.99% probability of being different). Such delay resulted in a reduction of exsheathment in RLE of 62% at 3 h, 38% at 6 h, and 12% at 9 h, relative to RL values. When PEG was added with the extract (RLE+PEG), the T50 (5.045 h) was similar to that of RL and RL+PEG. The IVRI method was adapted as an in vitro rumen exsheathment test (IVRET). The IVRET showed that H. contortus L3 exposed to G. floribundum extract delayed their exsheathment kinetics at different time points. The exsheathment delay was attributed to the polyphenol content of the extract.

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.

3′-Demethoxy-6-O-Demethylisoguaiacin and Norisoguaiacin Nematocidal Lignans from Artemisia cina against Haemonchus contortus Infective Larvae

Artemisia cina is a plant used in traditional Chinese medicine as a remedy for parasitic diseases. This study describes the isolation and chemical characterization of anthelmintic compounds of A. cina against Haemonchus contortus infective larvae (L3) through lethal testing. Previously, three extracts-n-hexane (HexAc), ethyl acetate (EtOAc) and methanol (MeOAc)-were evaluated at concentrations of 4 to 0.5 mg/mL, resulting in the HexAc extract with the greatest effect of 76.6% mortality of the larvae at 4 mg/mL. Then, this was chemically fractioned by polarity, obtaining seven fractions (C1F1-C1F7), and, when evaluated at concentrations from 2 to 0.25 mg/mL, the 2 mg/mL C1F5 fraction produced an effect against the nematode H. contortus of 100% mortality of the larvae. Thus, this fraction was fractionated again by column chromatography, obtaining twelve subfractions (C2F1-C2F12) which were evaluated from 1 to 0.125 mg/mL, with the C2F5 subfraction causing a nematicidal effect of 100% mortality. NMR analysis of one (1H, 13C and DEPT) and two dimensions (COSY, HSQC and HMBC) and mass spectrometry of this fraction allowed us to identify the mixture of 3'-demethoxy-6-O-demethylisoguaiacin and norisoguaiacin. Therefore, it can be assumed that the mixture of these compounds is responsible for the anthelmintic effect. These results indicate that A. cina containing anthelmintic compounds and might be used as an antiparasitic drug against H. contortus.

Identification of Somatic Proteins in Haemonchus Contortus Infective Larvae (L3) and Adults.

SummaryHaemonchus contortus is considered the most pathogenic nematode in sheep production systems based on grazing. Comparing infective larvae (L3) with adult parasites can lead to the identification of proteins that play an important role in parasite-host interactions. In this study, we report a list of H. contortus somatic proteins and made a comparative analysis of somatic proteins of L3 and adult worms. L3 and adult parasites were subjected to protein extraction and subsequently to peptide fractionation. Peptides were analysed by mass spectrometry and LC-MS/MS data analysis. Data analysis and search on SEQUEST and MASCOT against H. contortus from the WormBase ParaSite database resulted in the identification of 775 unique peptide sequences corresponding to 227 proteins at 1 % FDR. From these, 18 proteins were specific to L3 and 63 to adult parasites. The gene ontology (GO) enrichment analysis of the proteins specific to L3 and adult worms to gain insight into cellular components, molecular functions and biological processes that affect the parasite-host interaction showed some differences between the two parasite stages. The list of proteins found provides a database to identify target proteins that could be useful as biomarkers of the infection or in the generation of anthelmintic drugs that inhibit proteins essential for the establishment of the infection and the survival of adult parasites. They can also serve as new candidates for vaccine research.

Comparative study on pathological changes in sheep and goats experimentally infected with Haemonchus Contortus

BackgroundThe parasites causes localized and generalized infections to the host depends on the parasite attachment organs, depth of penetration, site of location and worm burdens.MethodsThe experimental study was done between December, 2019 and April, 2020 in the fly-proof experimental animal facility located in the premise of the College of Veterinary Medicine and Agriculture at Bishoftu, Ethiopia. A total of 14 male goat (G1 and G2) and 14 male sheep (G3 and G4) were allotted in to four equal groups. Single dose of 10,000 of infective larvae of Haemonchus contortus (L3) was orally administered to each animal in G1 and G3. Parameters such as body weight, PCV, haemoglobin, worm count, serum total protein, serum albumin, alkaline phosphatase and aspartate aminotransferase were measured. Moreover, histopathological sections were stained and examined for general changes as well as for changes in specific cells such as tissue eosinophilia and parietal cell population.ResultsThe findings show that 1) All infected sheep and goats developed the infection with higher mean worm burden in goats (5590) than sheep (2887) and the difference was significant (P < 0.05); 2) All infected sheep and goats exhibited a progressive anaemia; the level being more severe in goats than in sheep (P < 0.05) with mean PCVs of 13 and 18.6% respectively; 3) While body weight gain was minimal in sheep, goats have lost significant weight compared to pre-infection levels, to control animals or in relation to infected sheep (P < 0.05); 4) analysis of biochemical changes revealed marked reduction in serum total protein and albumin which was much more significant in goats than in sheep (P < 0.05); 5) the abomasum of infected sheep and goats have shown thickening, nodule development, eosinophilic infiltration and damage to parietal cells. Tissue eosinophilia was more prominent in sheep while parietal cell loss was severe in goats.ConclusionIn conclusion, goats under experimental infection and similar management condition with sheep develop much more severe infection and associated pathology compared to sheep and hence deserve special attention.

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.

Anthelminthic effects of extracts of indigenous browses from mid rift valley of Ethiopia

This study was conducted to evaluate the potential anthelminthic properties of extracts of leaves of indigenous browses (Acacia seyal, Acacia senegal, Acacia tortilis, Millettia ferruginea, and Vernonia amygadalina) based on three in vitro assays. Acetone extracts of browses at different concentrations (75 to 1200 μg/ml, for egg and larvae and 100mg/ml for an adult) were tested on three developmental stages of Haemonchus contortus (eggs, infective larvae, and adult worms) using egg hatch assay (EHA), larval migration inhibition assay (LMIA) and adult worm motility inhibition assay (AMIA). Significant effects were obtained with all five browses but differences were observed depending on the parasitic stages. The effects of five browse extracts on egg hatching were concentration-dependent, the highest (P&lt;0.05) egg hatch inhibition rate was observed at 1200 μg/ml concentration for all browses. All extracts had a higher effect (P&lt;0.01) than that of the negative control, phosphate buffer saline (PBS). In contrast, no concentration-response relationship was found for infective larvae and adult worms, although more potent effects were observed with the highest concentrations. The LMI rate (70%) induced by Vernonia amygadalina extract, at a concentration of 300 μg/ml, was the highest (P&lt;0.05) of all other browses, even at higher concentrations. The highest LMI rate (62%) induced by Acacia senegal extract at higher concentration, was lower than that of LMI rate (70%) induced by Vernonia amygadalina, at 300 μg/ml concentration. Vernonia amygadalina was found to be highly and rapidly effective against adult worms inducing the highest mortality rate (90%) as soon as 4 hrs after incubation. Overall, the in vitro results suggest that these five browses do possess anti-parasitic properties and Vernonia amygadalina showed the most effective anti-parasitic property. These effects remain to be confirmed through in vivo study.

Influence of faecal culture media and incubation time on the yield of infective larvae of Haemonchus contortus (Rudolphi 1803)

The aim of this experiment was to determine the yield of Haemonchus contortus third-stage larvae (L3) in faecal cultures in different conditions, including incubation time (7 or 14 days), the addition of inert additives (polystyrene pellets, vermiculite or no additive) and physical condition of the incubated faeces (ground or whole pellets). Twelve groups of 10 cultures each were arranged and incubated at 24 °C to evaluate the interaction of the above-mentioned conditions. Significantly, more L3 (p=0.0019 to p=0.0200) were recovered from cultures incubated for 7 days than for 14 days, except for the groups containing whole pellets with no additives (p=0.53) or with vermiculite (p=0.41). Larval yields from 7-day incubated cultures did not differ between groups (p=0.47), but for the whole pellets with vermiculite group, which yielded significantly less L3 (p<0.0001) than the rest of the cultures. Incubation for 14 days showed that cultures containing whole pellets with no additives yielded significantly more L3 (p<0.05) than the rest. Culturing faeces with H. contortus seems not to require inert additives or extra manipulation to obtain good L3 yields.

Protective efficacy of Barbervax® in Merino weaner sheep trickle infected with five doses of Haemonchus contortus infective larvae

Barbervax® protects sheep against H. contortus infection; however, the level of protection afforded by the vaccine at different levels of larval challenge under field conditions has not been reported. Therefore, this study was designed to investigate the protective efficacy of Barbervax® at increasing doses of trickle infection with H. contortus third stage larvae (L3). Merino lambs (220) were randomly allocated to challenge treatment groups and received Barbervax® vaccinations at approximately 8, 11, 15 and 22 weeks of age or not vaccinated (control group). Animals within each treatment group were subjected to one of five levels of H. contortus L3 trickle infection (0, 300, 600, 1200 and 2400 L3/week in two split doses). Trickle infections started two weeks after the third vaccination (week 0). Worm egg count (WEC), packed cell volume (PCV), antibody titre and bodyweight were measured at week 2 (start of trickle infection), week 5 and then every two weeks for 11 weeks. Vaccinated weaners had a significantly (P < 0.0001) lower faecal WEC than unvaccinated control animals. Vaccination induced significant specific antibody responses that were not influenced by level of trickle infection. Vaccination significantly reduced the number of weaners requiring salvage anthelmintic treatment (9.4% vaccinated weaners versus 33.6% unvaccinated). Vaccine protective index based on WEC was similar at all challenge levels (overall mean> 60%) and at the higher challenge levels did not reduce H. contortus infection to levels that would not require anthelmintic treatment. Therefore, it is suggested that under conditions of high larval challenge the use of Barbervax® should be integrated with other control methods.

Infective larvae of Haemonchus contortus found from the base to the top of the grass sward.

The resistance of gastrointestinal nematodes (GIN) of sheep to anthelmintic treatment has motivated researchers to seek alternatives to reduce the use of these drugs in sheep farming and decontaminate pastureland based on knowledge about the survival dynamics of larvae. The aim of this work was to evaluate the migration of the infective larvae (L3) of Haemonchus contortus at different times of the day, strata, and sward heights, with and without shade after the deposition of contaminated sheep feces. The grass species used here was Cynodon dactylon cv. Tifton 85 in four treatments: low sward height shade; low sward height sunshine; high sward height shade; and high sward height sunshine. The number of L3 recovered from the pasture at different times of the day did not differ. The highest number of L3 recovered was in shade. The number of L3 at different times and strata occurred uniformly, confirming that L3 remain in the same place after migrating from dung at the hottest times of the day. Infective larvae of H. contortus were able to migrate across all the strata regardless of the time of day in the summer season in humid subtropical climate.

Nematicidal Effect and Histological Modifications Induced by Hydrolysable Tannin Extract on the Third-Stage Infective Larvae of Haemonchus contortus.

Simple SummaryHaemonchus contortus is the most frequent and most important nematode parasite in the ruminants production of tropical and subtropical regions. The conduction of research to find resources that allow effective control of this parasitic disease, preferably focusing on green production, is necessary. Hydrolysable tannins (HT) are an important group of compounds synthetized by plants, some of them exhibit anti-cancer, antioxidant, anti-inflammatory, anti-ulcerative, or antiparasitic properties. We evaluated the effect of hydrolysable tannin extract (HTE) on larvae of H. contortus (L3). L3 were exposed to different HTE concentrations and times to analyze the mortality, and microscopically we explored physical damage on L3 caused by HTE. After 72 h of incubation with 2 mg/mL of HTE, we found a half of death larvae, and by microscopy we observed larvae destruction. Our results suggest that hydrolysable tannin extracted from chestnut could be considered as an alternative for parasitic control as a food additive in cattle.Haemonchus contortus is the most frequent and most important nematode parasite in the ruminants production of tropical and subtropical regions. There are strains resistant to all families of available anthelmintics. Consequently, the conduction of research to find other resources that allow effective control of this parasitic disease, preferably focusing on green production, is necessary. The aim of this study was to evaluate the effect of hydrolysable tannin extract (HTE) on larvae 3 (L3) of H. contortus in vitro. L3 were exposed to different HTE concentrations and times. In addition, both light and scanning electron microscopy were used to explore physical damage on L3 subjected to HTE activity. After 72 h of incubation, the mean lethal concentration of HTE was 2 mg/mL; this concentration has been previously referred to as safe for consumption in cattle. Scanning electron microscopy revealed H. contortus L3 destruction, damage was evident by separation of the sheath mainly in the cervical and caudal regions of the larva and by rupture of the cuticle with larval evisceration. Our results suggest that hydrolysable tannin extract from chestnut could be considered as a green alternative for parasitic control in ruminants.

Peripartum Phenomenon in Crioula Lanada Sheep Susceptible and Resistant to Gastrointestinal Nematodes.

This study aimed at evaluating parasitological and blood variables in native breed Crioula Lanada sheep belonging to the same herd, to identify and compare susceptible or resistant individuals to gastrointestinal nematodes during gestation and lactation phases. For this purpose, 18 Crioula sheep were used within 2 years of evaluation, in which blood and feces samples and weight of the animals were taken from their 4th month of gestation until the weaning of their lambs, in the 4th month postpartum. Feces samples were used for counting eggs per gram of feces (EPG) and, thus, to identify 12 resistant ewes (EPG < 1,000) and 6 susceptible (EPG > 1,000) to gastrointestinal nematodes. The identification of infective larvae was also performed. Blood was used for analysis of packed cell volume (PCV), eosinophil count, total plasma protein, and immunoglobulin G level against Haemonchus contortus infective larvae. The Kruskal–Wallis non-parametric comparison test was used to evaluate the differences between days of parturition and linear mixed-effects model using package lme4 in R to evaluate the groups. The main parasite species observed in the feces samples were Haemonchus, Trichostrongylus, Oesophagostomum, Ostertagia, and Cooperia in similar proportions in both groups. Susceptible ewes presented peaks of EPG at the beginning of lactation and lower PCV values throughout the study. No difference between groups was observed concerning other blood variables or body weight, but some changes were observed along with the gestation and lactation phases within each group. The physiological response of sheep to nematode infection is a useful tool to identify the most susceptible individuals within the same breed and herd and to select the most genetically resistant individuals.

Biological control of sheep nematode Haemonchus contortus using edible mushrooms

This study evaluated in vitro the morphology of ten strains of edible mushroom: Pleurotus ostreatus (strains 1123 and 0152), P. eryngii (strains 1290 and 1291), P. cornucopiae (strains 1328 and 1330), Coprinus comatus (strain 1103), Panus sp. (strain 801), Lentinula edodes (strain 401) and L. boryanus (strain 402) on different growing media. In addition, the in vitro anthelmintic activity (AH) of the mycelium of the fungi against Haemonchus contortus infective larvae was evaluated in Petri dishes. Finally, organic extracts of six of the fungi were synthesized and evaluated against H. contortus larvae. The highest mycelial growth rate was achieved by P. ostreatus (0152), Panus sp. (801), and P. ostreatus (1123) in wheat flour with agar (HIT). The highest biomass (1.43 g/Petri dish) was produced by Panus sp. 801 in the HIT medium. The highest percentages of mycelial AH mortality were caused by P. ostreatus 0152, P. cornucopiae 1328, P. ostreatus 1123, P. eryngii 1292 and L. edodes 401, at 88, 88.5, 91, 93 and 93.93% respectively. The biological activity of the fungi could be due to toxins, which inhibit the movement of nematodes and subsequently degrade them. The extracts that caused the highest mortality rates were: P. ostreatus 0152 (81.6%) and P. eryngii 1292 (90.8%) at 72 h following exposure to 200 mg/mL of extract. The results of the present study show that these fungi are candidates for future in vivo tests against H. contortus.

Long spelling periods are required for pasture to become free of contamination by infective larvae of Haemonchus contortus in a humid subtropical climate of São Paulo state, Brazil

The objective of this trial was to evaluate the period of spelling necessary for a pasture to become free of contamination by infective larvae of gastrointestinal nematodes (GIN) of sheep, in different seasons of the year, as well as to determine when the greatest pasture contamination occurs and how long it lasts. An area was divided into four paddocks, one for each season (spring, summer, autumn, and winter). In order to contaminate the paddocks with free living stages of GIN, eight ewes, naturally infected, grazed on each paddock for 14 consecutive days, starting on the following dates: autumn, on April 4, 2017; winter, on July 4, 2017; spring, on September 26, 2017; summer, on January 2, 2019. At the beginning and end of the grazing period, faecal samples were taken directly from the rectums of the ewes to count eggs per gram of faeces (EPG) and for faecal cultures. Every 14 days pasture samples were collected to assess the number of infective larvae (L3) per kilogram of dry matter. At the end of the 14 day ewe grazing period, 21 stakes were placed where there were faeces on the paddock. Subsequently, every 14 days, the faeces located at three of the stakes were collected and the L3 were recovered. After the exit of the ewes, monthly, two tracer lambs, free of helminth infection, were allocated into the paddock for 14 days. At the end of this period they were housed in covered stalls for 28 days. Faeces from the lambs were collected for individual EPG counting and faecal culture at 21 and 28 days after grazing. Infective larvae recuperation was observed from faeces and pasture in all seasons. In the autumn, spring, and summer, high EPG counts were observed in the first tracer lambs (8521, 4800, and 8064 EPG, respectively), while in winter, high infection (14132 EPG) of the animals was observed only from the second pair of tracer lambs. For a pasture to become “clean”, 322 days, 350 days, 294 days, and 182 days following contamination were necessary, respectively, in the autumn, winter, spring, and summer. In autumn, spring, and summer, massive contamination of the pasture with L3 occurred soon after an area had been grazed by infected sheep, while in winter this took a little longer. The contamination persisted, approximately, from a minimum of six months post contamination in summer to up to almost one year post contamination in winter.

In vitro effects of tannin and extracts of Bridelia ferruginea and Mitragyna inermis on the exsheathment of infective larvae of Haemonchuscontortus

ABSTRACT Bridelia ferruginea (Euphorbiaceae) and Mitragyna inermis (Rubiaceae) are two plants of the beninese pharmacopeia used in vivo for the control of gastrointestinal nematodes (GINs) in small ruminants. The objective of the present study is to explore the mechanism of bioactive compounds involved in the action of these two plants on the third-stage infective larvae (L3s) of Haemonchus contortus. Thus, sheathed L3s of H. contortus were incubated with acetone extracts of B. ferruginea and M. inermis at concentrations of 0, 150, 300, 600 and 1200 µg/mL for 3 h at 25°C. The L3s were then washed and artificially submitted to exsheathment in the presence of sodium hypochlorite solution. The role of tannins was verified by adding a tannin inhibitor, polyvinyl polypyrrolidone (PVPP), to the acetone extracts of these two plants for 2 h at 25°C. Acetone extracts from B. ferruginea and M. inermis inhibited the exsheathment of H. contortus larvae (p < 0.001) and this inhibitory effect was dose-dependent for M. inermis at the concentrations tested. Treatment of B. ferruginea and M. inermis extracts with PVPP was associated with a partial restoration of the exsheathment kinetics of H. contortus larvae (p < 0.001), confirming the predominant role of tannins but also the residual role of other secondary metabolites. These in vitro results suggest that these plants are endowed with anthelmintic (AHs) properties and therefore likely to be used as alternatives to synthetic molecules.

Nematocidal Effect of Oyster Culinary-Medicinal Mushroom Pleurotus ostreatus (Agaricomycetes) against Haemonchus contortus.

The nematocidal effect of Pleurotus ostreatus (white variety of oyster mushroom) aqueous extract (AE) was evaluated against Haemonchus contortus eggs and infective larvae (L3) in vitro and in artificially infected gerbils (Meriones unguiculatus). The chemical analyses indicated that constituents of AE are tridecanoic, tetradecanoic, linolelaidic, 9,15-octadecadienoic, and oxalic acids. P. ostreatus extract inhibited larval hatching by 100% at the concentration of 2.24 mg/mL and (50% effective concentration) EC50 of 0.73 mg/mL. In the larval development test, AE induced a larvicidal effect at the concentration of 50 mg/mL and EC50 of 17.24 mg/mL. The larval migration test revealed a reduction of 94.7% at a concentration of as low as 4 mg/mL and EC50 of 1.25 mg/mL. No significant effects of treatment with P. ostreatus AE were seen on H. contortus in the gerbil model. Thus, our results demonstrate an important nematocidal in vitro effect of P. ostreatus AE against the parasite H. contortus. However, further investigations are necessary to confirm the anthelmintic potential of P. ostreatus extract in small ruminants.

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

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. 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. Anthelmintic activity was assessed using the egg hatching inhibition assay (EHI) and mortality tests. A complete hydroalcoholic extract (HA-E) at 12.5-50mg/mL, an aqueous fraction (Aq-F) at 3.12-25mg/mL and an ethyl acetate fraction (EtOAc-F) at 3.12-25mg/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 0.62-5mg/mL concentrations. In addition, thiabendazole (0.6mg/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. The EtOAc-F fraction showed the highest ovicidal and larvicidal activities, at close to 100% at 3.12 and 6.25mg/mL, respectively. The treatments C1F2, C1F3 and C2F3 displayed the main ovicidal activity (80-100%) at 2.5mg/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. 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.

The Use of Melastoma malabatricum and Manihot esculenta extract as natural anthelmintic on the Performance of Kacang goat

This study was carried out to evaluate anthelmintic activity of the combination Melastoma malabatrucum and Cassava (Manihot esculenta) leaves extract on the performance of Kacang goat The first experiment was conducted to examine the efication of thus extract on larva development assay and adult motility assay. In the second experiment, twenty Kacang goat were distributed into four groups according to a Completely Randomized Design namelly P1= 75% Melastoma malabatricum extract + 25% Manihot esculenta extract. P2= 50% Melastoma malabatricum extract + 50% Manihot esculenta extract P3= 25% Melastoma malabatricum extract + 75% Manihot esculenta extract P4= : Single dose Ivermectin as positif control. Extract All experimental goats were dewormed with Albendazole and treated prophylactically with a broad-spectrum antibiotic before the experiment. Goat were infected orally with 200 infective Haemonchus contortus larvae (L3). Result indicated that Melastoma malabatrucum leaves extract inhibited larvae development assay by 77% compared with 71.5% on Cassava (Manihot esculenta) leaves extract. For the mean of adult motility assay increase in the concentration of extract from 3.125-50 mg/mL. Our finding also found the was significant different (P<0.05) on dry matter intake and there no significant different on live weight, no mortality was found during experimental periods.