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.

Evaluation of a combination of Citrus aurantium var. Dulcis essential oil and albendazole for the treatment of sheep gastrointestinal nematodes.

Citrus fruits are consumed all over the world and their by-products are used for animal feed and essential oils production. This study aimed to evaluate the in vitro and in vivo activity of Citrus aurantium var. Dulcis essential oil (CaEO) combined with ABZ against benzimidazole resistant Haemonchus contortus. In vitro egg hatching assays (EHA) were performed using CaEO and ABZ to estimate the effective concentration to achieve 50% egg death (EC50) values and calculate the test essential oil and drug combinations using a simplex-centroid mixture design. These concentrations were used for a second round of EHAs. Sixteen sheep were randomly allocated into two groups and treated with ABZ and the combination of CaEO and ABZ, and faecal egg count reduction tests were performed. In the first round of EHA, CaEO and ABZ showed EC50 values of 0.57 and 0.0048 mg mL-1, respectively. The H. contortus strain used in the study was shown to be highly benzimidazole resistant, with only 1.5% of parasites having susceptible ß-tubulin SNP genotypes. The ABZ reduced the shedding of nematode eggs by 78%, however, its combination with CaEO reduced faecal egg counts by only 9%. The present study is important to highlight the interferences of natural products in anthelmintic metabolism and consequently in drug efficacy.

Computed tomography and radioactive 32P detected phosphorus impairment in metabolism, reduced bones density and animal performance caused by mixed infection of Haemonchus contortus and Trichostrongylus colubriformis in sheep.

Phosphorus (P) is one of the main minerals present in the animal body and exerts crucial functions in the organism. P is present at all cell membranes and integrates the structure of bones, being necessary its supplementation in ruminants due to the deficiency of this mineral in the pastures. One of the principal factors that compromise its metabolization are gastrointestinal nematodes (GIN). Thus, the objective of this study was evaluate the performance and metabolism of P through its distribution in the animal body, density of bones and muscles, dynamic fluxes, biological availability and half live of P, concentration of P in tissues and bones of lambs simultaneously infected with the most prevalent GIN to sheep, in tropical or subtropical areas, (Haemonchus contortus and Trichostrongylus colubriformis) using the isotopic dilution technique with 32P radioisotope. Twenty Santa Ines sheep with seven months of age and averaging initial weight of 30.8 ± 6.41 kg were used and allocated to one of two treatments. Ten animals were orally infected (a single dose of 30,000 L3 larvae of T. colubriformis + 10,000 L3 larvae of H. contortus), and ten animals were not infected (control group). During the experimental, samples of blood, feces, urine, and diet refusals were collected and weighting were performed. A computed tomography was performed twice, before infection and at the end of the experiment, to evaluate changes in body composition. On 64-d after experimental infection, animals received an intravenous injection of 32P solution, and 7-d after they received radioisotope injection. The experimental animals were slaughtered, and tissue and bones were collected for P concentrations. The results showed that the parasitic infection compromised the absorption of P, impairing the metabolism, decreasing the mineral bioavailability increasing P bones reabsorption, and reducing bones density, also negatively compromising the infected animal performance.

Phytochemical profile and anthelmintic effects of Laurus nobilis essential oil against the ovine nematode Haemonchus contortus and the murine helminth model Heligmosomoides polygyrus.

Small ruminant production in tropical and temperate countries faced substantial anthelmintic resistance due to the intensive use of commercial anthelmintic drugs. Therefore, alternative treatments including natural bioactive compounds with anthelmintic potential have been investigated looking for its successfully use in the parasite control. In the present study, we describe the chemical profile of Laurus nobilis essential oil (EO), the in vitro anthelmintic activity of L. nobilis EO against Haemonchus contortus and its in vivo anthelmintic effect against the murine helminth parasite model Heligmosomoides polygyrus. Chromatographic profile of L. nobilis (EO) extracted from the leaves of L. nobilis have shown the presence of monterpens 1,8-cineol (Eucalyptol) (29.47%), D-Limonène (18.51%) and Linalool (10.84%) in high fractions. The in vitro anthelmintic potential was expressed by an ovicidal effect against H. contortus egg hatching with inhibition value of 1.72 mg/mL and 87.5% of immobility of adult worms after 8 h of exposure to 4 mg/mL of L. nobilis EO. Regarding, the in vivo anthelmintic potential, L. nobilis (EO) at 2400 mg/kg bw completely eliminated the egg output of H. polygyrus after 7 days of oral treatment, together with a 79.2% of reduction in total worm counts. Based on the obtained results, L. nobilis EO showed promising in vitro and in vivo anthelmintic capacities against gastrointestinal parasites.

In vitro and in vivo anthelmintic properties of Caesalpinia coriaria fruits against Haemonchus contortus.

The purpose of the present study was to assay the in vitro and in vivo anthelmintic activity (AA) of Caesalpinia coriaria (Cc) mature fruits against the nematode Haemonchus contortus (Hc). The Hc infective larvae were used to assess the in vitro AA through larval mortality assay. The exposure of larvae to the different treatments was performed in 96-well microtitration plates. The treatments were as follows: hydroalcoholic extract (HA-E, at 25-100 mg/mL), aqueous fraction (Aq-F, at 12.5-50 mg/mL), organic fraction (EtOAc-F at 12.5-50 mg/mL), compounds (1, methyl gallate and 2, gallic acid at 1.25-10 mg/mL), positive control (ivermectin at 5 mg/mL) and two negative controls (distilled water and 4% methanol). After exposure, dead and live larvae were quantified and results were compared to their controls. The in vivo assay was carried out by a faecal egg count reduction test (FECRT); artificially infected goat kids (F1: Boer x Nubia) were treated with Cc ground dried fruits to assess the AA. The treatments were established as follows: G1-untreated goats (negative control), G2-goats dewormed with ivermectin (positive control), G3-goats fed with Cc mature fruits (10% of their diet). Results in both in vitro and in vivo assays were analysed using an ANOVA through random design, applying a general linear model and mixed models. The in vitro results showed an evident larvicidal effect of the HA-E, EtOAc-F from Cc, indicating that the compound responsible for the AA was gallic acid. The results of the in vivo study corroborated the anthelmintic properties of Cc, reaching 78.6% reduction in the elimination of Hc eggs per gram of faeces. This plant represents a potential natural anthelmintic for the control of haemonchosis in goats under grazing conditions. Future studies should standardise the Cc extract or dried fruits for use in the management of nematodiasis in goat herds.

In vitro anthelmintic activity of an R-carvone nanoemulsions towards multiresistant Haemonchus contortus.

This work aimed to evaluate the in vitro anthelmintic effect of carvone nanoemulsions on Haemonchus contortus. Three R-carvone nanoemulsions were prepared: uncoated R-carvone nanoemulsions homogenized in a sonicator (UNAlg-son) and homogenized in an ultrahomogenizer (UNAlg-ultra) and sodium alginate-coated R-carvone (CNAlg-ultra). The physicochemical characterizations of the nanoemulsions were carried out. The anthelmintic activity was evaluated using egg hatch test (EHT), larval development test (LDT) and adult worm motility test (AWMT). Changes in cuticle induced in adult H. contortus were evaluated by scanning electron microscopy (SEM). The results were subjected to analysis of variance and compared using the Tukey test (P < 0.05). The effective concentration to inhibit 50% (EC50) of egg hatching and larval development was calculated. The particle sizes were 281.1 nm (UNAlg-son), 152.7 nm (UNAlg-ultra) and 557.8 nm (CNAlg-ultra), and the zeta potentials were −15 mV (UNAlg-son), −10.8 mV (UNAlg-ultra) and −24.2 mV (CNAlg-ultra). The encapsulation efficiency was 99.84 ± 0.01%. SEM of the nanoemulsions showed an increase in size. In EHT, the EC50 values of UNAlg-son, UNAlg-ultra and CNAlg-ultra were 0.19, 0.02 and 0.17 mg mL−1, respectively. In LDT, they were 0.29, 0.31 and 0.95 mg mL−1 for UNAlg-son, UNAlg-ultra and CNAlg-ultra, respectively. The adult motility inhibition was 100% after 12 h of exposure to UNAlg-ultra and CNAlg-ultra, while for UNAlg-son, it was 79.16%. SEM showed changes in the buccal capsule and cuticular damage. It was concluded that R-carvone nanoemulsions showed antiparasitic action demonstrating promise for the control of infections caused by gastrointestinal nematodes in small ruminants.

Eucalyptus oil nanoemulsions against eggs and larvae of Haemonchus contortus.

Haemonchus contortus is a highly pathogenic and prevalent helminth that causes many deaths in sheep herds. Anthelmintics are usually employed to overcome this issue; however, they do not guarantee immediate and lasting efficacy because of the occurrence of drug-resistant parasites. Among substances that are used in scientific studies for parasitic control, essential oils are known to have different pharmacological properties. However, they demonstrate instability owing to several factors, and therefore, nanoemulsification is considered an alternative to control the instability and degradability of these compounds. The objective of this study was to evaluate the cytotoxicity of nanoemulsions containing essential oil of Eucalyptus globulus against the blood of healthy sheep and to verify their activity against the parasite H. contortus in sheep. The results presented adequate nanotechnological characteristics (diameter 72 nm, PDI 0.2, zeta -11 mV, and acidic pH) and adequate morphology. Further, the corona effect and cytotoxic profiles of the free oil and nanoemulsion against blood cells from healthy sheep were evaluated. The tests results did not present a toxicity profile. For evaluating efficacy, we observed an important anthelmintic action of the nanoemulsion containing oil in comparison to the free oil; the results demonstrate a potential role of the nanoemulsion in the inhibition of egg hatchability and the development of larvae L1 to L3 (infective stage). Based on these results, we developed an important and potential anthelmintic alternative for the control of the parasite H. contortus.

An Artemisia cina n-hexane extract reduces the Haemonchus contortus and Teladorsagia circumcincta fecal egg count in naturally infected periparturient goats.

The objective of this study was to evaluate an n-hexane extract of Artemisia cina (Acn-h) as a natural anthelmintic treatment for periparturient goats naturally infected with the nematodes Haemonchus contortus and Teladorsagia circumcincta. A total of 200 periparturient Alpine and Nubian goats were used. Deworming criteria were based on the following parameters: fecal egg account (epg), ocular mucosa color (OMC), and body condition (BC). A previous analysis using coprocultures of the flock revealed the presence of H. contortus (80%) and T. circumcincta (20%). The Acn-h contained two new compounds identified by mass spectrometry data as isoguaiacin and norisoguaiacin at 284.14 and 315 m/z. The lethal effects of Acn-h at 0.5, 1, 2, and 4 mg/mL on H. contortus adult stages were 31.6, 66.5, 81.3, and 86.9%, respectively (p < 0.05), showing similar efficacy at 2 and 4 mg/mL with albendazole (positive control group). Then, two experimental groups, with 100 goats in peripartum in each, were distributed randomly and treated at day 0 as follows: group 1 = 4 mg/kg of Acn-h as single oral dose, and group 2 = control group, treated with water (as a placebo). The epg, OMC, and BC parameters were recorded at 0 (periparturient period), 7 (birth period), and 23 (postpartum) days and analyzed using a completely randomized design with Duncan's test for comparison of means and analysis of variance. The following epg reductions were recorded in the Acn-h-treated group as follows: 20.1 ± 34.4 and 31.7 ± 38.2% at days 7 and 23 compared to the control group. During the whole experiment, no significant differences in OMC or BC were observed in relation to the control group, excepting at day 23 (p < 0.05) for BC in the group treated with A. cina. Thus, Acn-h can be a useful natural alternative tool for the control of the nematodes H. contortus and T. circumcincta in periparturient goat flocks.

Effects of Ferula asafetida, closantel, albendazole, oxfendazole, and ivermectin against Haemonchus contortus in goats and sheep.

BACKGROUND: Haemonchosis is a fatal disease of small ruminants caused by the parasite Haemonchus contortus (H. contortus). The most common drugs used in the treatment of H. contortus include albendazole, oxfendazole, and ivermectin. However, as previously reported in the treatment of haemonchosis, these medicines have acquired drug resistance problems over time. Interestingly, natural plant compounds have demonstrated promising effects in the treatment of H. contortus. Therefore, the current study evaluated the effects of plant extract, Ferula asafetida, against common drugs such as albendazole, oxfendazole, ivermectin, and closantel for the treatment of haemonchosis in small ruminants. METHODOLOGY: The current study was conducted on different small ruminant farms in Kasur District, Punjab, Pakistan. The positive animals (n = 720) after coprological examination were selected in this study and divided into two major groups (n = 360 goats and n = 360 sheep). Further, animals were divided into five treatment groups (A-E) and one control group with no treatment (F). Albendazole, oxfendazole, ivermectin, closantel, and Ferula asafetida were administered orally to groups A-E, respectively. The eggs per gram feces (EPG) were determined through the McMaster technique on days 0, 7th, and 14th of treatment. RESULTS: The results showed a significantly higher efficacy of closantel and Ferula asafetida against H. contortus in both goats (100% and 70%; p < 0.05) and sheep (99% and 87%; p < 0.05), respectively. No correlation was observed between EPG reduction with age and gender in both goats and sheep. CONCLUSION: Allopathic drug closantel and herbal drug, Ferula asafetida, have been proved an effective dewormer against H. contortus in small ruminants.

Can orange essential oil reduce the severity of parasitic infection in sheep?

In this study, a three-step experiment was conducted to evaluate the effectiveness of orange essential oil (OEO) to reduce the severity of parasitic infection in lambs naturally infected with gastrointestinal parasites. In the first and second stages of the experiment, the lambs were distributed in groups (n = 10): without treatment (control), treated with levamisole (7.5 mg/kg), and treated either with 200, 300 or 400 mg/kg OEO in the first stage and with two doses of 300 or 350 mg/kg each of OEO with a six-hour interval in the second stage. In the third stage of the experiment, 20 lambs were distributed into control and treated groups (two doses of 350 mg/kg OEO) and slaughtered for determining the parasite count in the abomasum and small intestine. Evaluations were performed on D0, D3, D7, D10, and D14 by determining the egg count per gram of feces (EPG), and the percentage of EPG reduction was calculated in comparison to D0. Coproculture was performed on days D0 and D14 of the first and second stages of the study. In the first stage, the EPG increased by 20.90 and 100.99% and decreased by 5.54% on D14 in the groups treated with 200, 300, or 400 mg/kg OEO, respectively. At the second stage, the EPG of the OEO groups decreased by more than 96% on D3, but on D14 was decreased by 0.82% and 77.07% in the 2 × 300 and 2 × 350 mg/kg groups, respectively. Haemonchus sp. was the highest percentage of collected larvae in all coproculture tests, ranging from 90 to 100%. In the third stage, there was no difference (P > 0.05) in the mean number of parasites between the OEO groups and the control. Although the decrease in EPG of more than 96% on D3 did not persist in the other evaluations, and immature forms and adult parasites were collected at slaughter, the EPG reduction of 77.07% on D14 with the dose of 700 mg/kg (OEO2 × 350) suggests that OEO can help to reduce the severity of parasitic infection and, consequently, to diminish pastures contamination, when used together with other methods to control gastrointestinal parasites in sheep.

Anthelmintic activity of methanol extract of Dennettia tripetala G. Baker (Annonaceae) fruits against Haemonchus contortus in red Sokoto goats.

Haemonchosis is a gastrointestinal parasitic disease of economic importance in ruminants especially sheep and goats. In view of the rising costs of conventional veterinary anthelmintics and the development of resistance by Haemonchus contortus, there is a need to develop alternative ethnoveterinary therapies for the treatment and management of Haemonchosis. This study investigated the anthelmintic activity of Dennettia tripetala G. Baker (Annonaceae) fruits against Haemonchus contortus in red Sokoto goats. The maximum tolerated dose was determined in adult albino rats administered, 3000, 4000, and 5000 mg/kg body weight of the crude methanol extract (CME) fraction, and observed over a period of 48 h for signs of toxicity and mortality. The in vivo anthelmintic activity was evaluated using 20 kids infected with H. contortus and randomly allocated into 5 groups (1, 2, 3, 4, and 5). Kids in groups 1, 2, and 3 were treated with CME at doses of 1250, 2500, and 5000 mg/kg respectively for 3 consecutive days per os. Groups 4 and 5 were treated with albendazole (7.5 mg/kg) once and distilled water (5 ml) respectively and served as treated and untreated controls. Blood samples were collected for haematology. The maximum tolerated dose of the CME of D. tripetala did not produce observable signs of toxicity or death in all the rats given up to 5000 mg/kg. There was significant (P < 0.05) reduction in faecal egg count by CME at doses of 1250 mg/kg (91.6%), 2500 mg/kg (98.5%), and 5000 mg/kg (100%) at day 14 post-treatment. The total plasma protein (TPP) and FAMACHA© scoring values were significantly improved (P < 0.05) in the treated groups. These results indicate that methanol extract and fractions of D. tripetala fruits possess beneficial anthelmintic activity against H. contortus and may be a suitable alternative anthelmintic candidate for the control of haemonchosis in goats.

The ATP bioluminescence assay: a new application and optimization for viability testing in the parasitic nematode Haemonchus contortus.

The parasitic gastrointestinal nematode Haemonchus contortus causes serious economic losses to agriculture due to infection and disease in small ruminant livestock. The development of new therapies requires appropriate viability testing, with methods nowadays relying on larval motility or development using procedures that involve microscopy. None of the existing biochemical methods, however, are performed in adults, the target stage of the anthelmintic compounds. Here we present a new test for the viability of H. contortus adults and exsheathed third-stage larvae which is based on a bioluminescent assay of ATP content normalized to total protein concentration measured using bicinchoninic acid. All the procedure steps were optimized to achieve maximal sensitivity and robustness. This novel method can be used as a complementary assay for the phenotypic screening of new compounds with potential antinematode activity in exsheathed third-stage larvae and in adult males. Additionally, it might be used for the detection of drug-resistant isolates.

Performance of lambs under four levels of dietary supplementation and artificially mix-infected with Haemonchus contortus and Trichostrongylus colubriformis.

The effect of four diets on the performance of Dorper lambs that were artificially mix-infected with 1000 infective larvae (L3) of Haemonchus contortus and 1000 L3 of Trichostrongylus colubriformis every three days for 12 weeks was evaluated. For each diet, one infected group (n = 7) and one control group (n = 4) were set up. The four diets contained the following proportions of hay (H) and concentrate (C): Diet 1 - 100%H; Diet 2 - 75%H:25%C; Diet 3 - 50%H:50%C; Diet 4 - 25%H:75. All the infected groups showed decreases in mean packed cell volume (PCV) and total plasma protein, especially the Diet 1-infected, which was also the group with the highest fecal egg count. The diets and infection had significant effects (P < 0.05) on daily body weight gain (DBWG), cold carcass weight and daily food consumption. The control and infected groups had mean DBWG (in kg), respectively, of 0.048 and 0.025 with Diet 1; 0.082 and 0.067 with Diet 2; 0.188 and 0.189 with Diet 3; and 0.303 and 0.221 with Diet 4. In conclusion, the infection affected the productive performance regardless of the diet; however, with increased metabolizable protein and energy, the risk of occurrence of anemia and hypoproteinemia diminished.

In Vitro Nematocidal Effect and Anthelmintic Activity of Artemisia cina Against Haemonchus contortus in Gerbils and Relative Expression of Hc29 Gene in Transitional Larvae (L3-L4).

PURPOSE: (1) To assess the in vitro activity of Artemisia cina against Haemonchus contortus L3 (HcL3) and in transitional (L3-L4) larvae (HcTrL3-L4); (2) to quantify the relative expression of the Hc29 gene in HcTrL3-L4 exposed to the A. cina n-hexane extract; and (3) to assess the anthelmintic activity (AA) of the A. cina organic extracts in gerbils artificially infected with H. contortus (HcArt/inf/gerbs). METHODS: The in vitro assay was carried out in 96-well microtitration plates. The following A. cina extracts: ethyl acetate (Ac-EtOAcEx), n-hexane (Ac-n-HexEx), and methanol (Ac-MethEx) were assessed at 1 and 2 mg/mL against HcL3 and HcTrL3-L4 at 24 h exposure. Relative expression of the Hc29 gene in HcTrL3-L4 was obtained by RT-PCR. For assessing the AA, six groups of five HcArt/inf/gerbs were used. Groups were treated orally with 4 mg/kg BW of A. cina extracts. Five days after treatment, the gerbils were necropsied and nematodes counted. RESULTS: The highest in vitro activities (75 and 82.6%) were shown by Ac-n-HexEx at 1 and 2 mg/mL, respectively. For HcTrL3-L4 the highest in vitro activities (69 and 23%) were shown by Ac-n-HexEx and isoguaiacine at 0.625 mg/mL, respectively. Also, upregulation of H. contortus Hc29 gene by 13- and 80-fold (p < 0.01) was observed on the HcTrL3-L4 stage after exposure to Ac-n-HexEx extract and isoguaiacine at 0.078 mg/mL, respectively. Reduction percentage was 100% in HcArt/inf/gerbs treated with Ac-n-HexEx. CONCLUSIONS: We conclude that the Ac-n-HexEx and isoguaiacine compound had anthelmintic efficacy against H. contortus and L3 and HcTrL3-L4.

Nematicidal activity of a hydroalcoholic extract of the edible mushroom Neolentinus ponderosus on L3 larvae of Haemonchus contortus.

OBJECTIVE: In the present study, the in vitro and in vivo effect of the hydroalcoholic extract of Neolentinus ponderosus (EHNP) on L3 larvae of Haemonchus contortus was evaluated. MATERIAL AND METHODS: The N. ponderosus fungus was cultivated in potato dextrose liquid medium for 7 days at 120 rpm and 25 °C. Subsequently, the EHNP was obtained; in vitro bioassays were performed in 96-well plates. Furthermore, an in vitro confrontation with different concentrations of EHNP was carried out at 72 h against L3 larvae of H. contortus. The controls used were distilled water and ivermectin at 5 mg/mL. Subsequently, the in vivo activity of EHNP was evaluated using the gerbil against H. contortus L3 larvae as an experimental model. The experimental design consisted of four groups with: (1) distilled water, (2) fenbendazole at 20 mg/kg of body weight, (3) EHNP at a dose of 81 µg/mL, and (4) EHNP at a dose of 40 µg/mL. RESULTS: In vitro study showed 97% mortality of the parasite H. contortus at a concentration of 3.4 mg/mL and a lethal concentration (LC90) of 2 mg/mL EHNP. In the in vivo assessment the highest mortality was (49%) at 72 h at a concentration of 81 µg/mL bw. CONCLUSION: The result of the present study shows that EHNP has nematicidal activity in vitro and in vivo tests (close to 97% and 50%, respectively), the fungus N. ponderosus should be considered in future tests to elucidate the secondary metabolites through spectroscopic studies.

Inhibition of Protease and Egg Hatching of Haemonchus contortus by Soybean Seed Exudates.

Gastrointestinal nematode infection of small ruminants causes losses in livestock production. Plant compounds show promises as alternatives to commercial anthelmintics that have been exerting selective pressures that lead to the development of drug-resistant parasites. Soybean (Glycine max) is an economical value crop, with a higher protein content compared to other legumes. The objective of this study was to evaluate whether the protease inhibitors exuded from the G. max mature seeds have anthelmintic activity against Haemonchus contortus. To obtain the soybean exudates (SEX), mature seeds were immersed in 100 mM sodium acetate buffer, pH 5.0, at 10 C, for 24 hr. Then the naturally released substances present in SEX were collected and exhaustively dialyzed (cutoff 12 kDa) against distilled water. The dialyzed seed exudates (SEXD) were heated at 100 C for 10 min and centrifuged (12,000 g, at 4 C for 15 min). The supernatant obtained was recovered and designated as the heat-treated exudate fraction (SEXDH). The protein content, protease inhibitor activity, and the effect of each fraction on H. contortus egg hatch rate were evaluated. The inhibition extent of SEX, SEXD, and SEXDH on H. contortus egg proteases was 31.1, 42.9, and 63.8%, respectively. Moreover, SEX, SEXD, and SEXDH inhibited the egg hatching with EC50 of 0.175, 0.175, and 0.241 mg ml-1, respectively. Among the commercial protease inhibitors tested, only EDTA and E-64 inhibited the H. contortus hatch rate (79.0 and 28.9%, respectively). We present evidence demonstrating that soybean exudate proteins can effectively inhibit H. contortus egg hatching. This bioactivity is displayed by thermostable proteins and provides evidence that protease inhibitors are a potential candidate for anthelmintic use.

Performance of lambs under four levels of dietary supplementation and artificially mix-infected with Haemonchus contortus and Trichostrongylus colubriformis / Efeito de quatro dietas na performance de cordeiros artificialmente infectados com Haemonchus contortus e Trichostrongylus colubriformis

Abstract The effect of four diets on the performance of Dorper lambs that were artificially mix-infected with 1000 infective larvae (L3) of Haemonchus contortus and 1000 L3 of Trichostrongylus colubriformis every three days for 12 weeks was evaluated. For each diet, one infected group (n = 7) and one control group (n = 4) were set up. The four diets contained the following proportions of hay (H) and concentrate (C): Diet 1 - 100%H; Diet 2 - 75%H:25%C; Diet 3 - 50%H:50%C; Diet 4 - 25%H:75. All the infected groups showed decreases in mean packed cell volume (PCV) and total plasma protein, especially the Diet 1-infected, which was also the group with the highest fecal egg count. The diets and infection had significant effects (P < 0.05) on daily body weight gain (DBWG), cold carcass weight and daily food consumption. The control and infected groups had mean DBWG (in kg), respectively, of 0.048 and 0.025 with Diet 1; 0.082 and 0.067 with Diet 2; 0.188 and 0.189 with Diet 3; and 0.303 and 0.221 with Diet 4. In conclusion, the infection affected the productive performance regardless of the diet; however, with increased metabolizable protein and energy, the risk of occurrence of anemia and hypoproteinemia diminished.

Resumo Avaliou-se o efeito de quatro dietas sobre o desempenho de cordeiros Dorper infectados artificialmente, a cada três dias, durante 12 semanas, com 1.000 larvas infectantes (L3) de Haemonchus contortus e 1.000 L3 de Trichostrongylus colubriformis. Um grupo infectado (n=7) e um controle (n=4) foram constituídos para cada uma das dietas, que continham a seguinte proporção de feno (F) e concentrado (C): Dieta 1 - 100%F; Dieta 2 - 75%F:25%C; Dieta 3 - 50%F:50%C; Dieta 4 - 25%F:75%C. Todos os grupos infectados apresentaram redução nas médias de hematócrito (VG) e proteína plasmática total (PPT), principalmente o grupo Dieta 1 infectado, que também apresentou maior contagem de ovos nas fezes. Houve efeito significativo da dieta e da infecção (P <0,05) no ganho em peso médio diário, peso da carcaça e consumo diário de alimento. Os grupos controle e infectado tiveram ganho em peso médio diário (em kg), respectivamente, de 0,048 e 0,025 na Dieta 1; 0,082 e 0,067 na Dieta 2; 0,188 e 0,189 na Dieta 3; e 0,303 e 0,221 na Dieta 4. Concluiu-se que a infecção afetou o desempenho produtivo independentemente da dieta, porém, com o aumento da proteína e da energia metabolizável, o risco de ocorrência de anemia e hipoproteinemia diminuiu.

Ovine haemonchosis: a review.

Sheep farming is the backbone of a rural economy in developing countries, and haemonchosis is a major impediment in the way of its progress. Haemonchus contortus (H. contortus) infection persists all over the world particularly in the tropical and sub-tropical regions. Various review articles have been published to substantially cover one or more aspects of its morphology, prevalence, pathogenesis, symptoms, diagnosis, immune response, drug resistance, treatment, and control measure. The objective of this paper is to briefly review past and present information available in the aforementioned areas in one place to enable the readers to fully understand the problem from a broader perspective. H. contortus parasite harbours in abomasum of affected animal and feeds on its blood, producing mild to severe symptoms and even death in acute form. The parasite thus inflicts heavy production losses and is of economic importance. H. contortus has developed diverse characters over the years leading to limited success in the production of vaccines. Indiscriminate use of the anthelmintics has produced drug resistance against almost all conventional products. Efficacy of medicinal plants and non-conventional chemicals has been reported under controlled experiments; however, research on their adverse effects on growth and fertility is yet to be studied. Research on molecular tools for identification and introduction of resistant genes into the flock is also underway but still a long journey to find its field application. Crossbreeding may compromise the production traits of the existing flock. In given circumstances, a targeted selective treatment approach along with selective breeding, culling of more susceptible animals, and maintaining a good body condition score through the provision of a balanced diet remains a workable strategy to control haemonchosis in sheep.

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.

Chemical characterization and in vitro biological activity of Cymbopogon citratus extracts against Haemonchus spp. and Trichostrongylus spp. nematodes from sheep.

Medicinal plants have been the focus of several studies due to their nematicide properties which can be used to control nematodes in sheep. No study has examined the morphological effects of Cymbopogon citratus on nematodes. Thus, this study evaluated the chemical composition, nematicidal activity and effects of C. citratus extracts on the morphology of eggs and infective larvae (L3) of sheep. Aqueous and methanolic extracts and fractions of C. citratus were obtained and analysed in vitro. The C. citratus extracts were effective against Haemonchus spp. and Trichostrongylus spp. larvae and eggs. Ten fractions were obtained from C. citratus, six of which had high ovicidal activity at 1000 µg mL-1, and two fractions had high activity at all tested concentrations. The phytochemical analysis identified the presence of compounds such as terpenoids, various ketones, esters, and fatty acids. The ultrastructural analysis showed deformations of the cuticle and wilting along the body of the nematodes at all concentrations. The muscular layer, intestinal cells and the mitochondria profile showed damage compared to the typical pattern. Ultra-thin sections of eggs treated with methanolic fractions of C. citratus presented modifications. This study showed the biological activity and effects of C. citratus on the gastrointestinal nematodes in sheep.