Triclabendazole resistance in Fasciola hepatica: First report in sheep from the Santa Cruz province, Argentinian Patagonia.

In the fall of 2022, decreased triclabendazole (TCBZ) efficacy against F. hepatica was suspected in a sheep farm located in the Santa Cruz province, Argentinian Patagonia. Since TCBZ-resistance in F. hepatica has never been reported in this province, this study aimed to confirm potential TCBZ-resistance in F. hepatica and to evaluate the efficacy of closantel (CLO) and nitroxinil (NTX), through faecal egg count reduction test (FECRT), and the efficacy of albendazole (ABZ) through the in vitro egg hatch test (EHT) in sheep. Sixty-eight (68) animals were selected from a herd of eighty (80) female Merino naturally infected with F. hepatica based on eggs per gram of F. hepatica (EPGFh) counts and assigned into four (4) groups (n = 17 per group): Group Control, animals did not receive anthelmintic treatment; Group TCBZ, animals were orally treated with TCBZ (12 mg/kg); Group CLO, animals were orally treated with CLO (10 mg/kg); and Group NTX, animals were subcutaneously treated with NTX (10 mg/kg). The fluke egg output was monitored on days 0 and 21 post-treatment. For the EHT, liver fluke eggs were isolated from faecal samples (approx. 50 g) collected from animals of the control group. TCBZ efficacy against liver fluke was 53.4%, confirming the presence of TCBZ-resistant isolates on the farm. CLO and NTX were highly effective (100%) for the treatment of F. hepatica on this farm. The EHT was carried out in two different laboratories, in which was observed an ABZ efficacy of 95.8 (Bariloche) and 96.5% (Tandil). These results indicate the ABZ susceptibility of this F. hepatica isolate and the inter-laboratory precision of the test.

Chemical Composition, Anthelmintic Activity, and Mechanism of Action of Lippia dominguensis Mold. Essential Oil on Haemonchus contortus.

Gastrointestinal nematode parasitism is a major burden to small ruminant production globally, compounded by increasing anthelmintic resistance. Previous studies have identified essential oils (EOs) from the Lippia genus with antiprotozoal and anthelmintic effects. Lippia dominguensis Moldenke (Ld), an endemic specie from the Dominican Republic, has similar popular uses, however, is chemically and pharmacologically yet uncharacterized. Here, we investigated the in vitro anthelmintic activity of LdEO and its ultrastructural effects on eggs and adult nematodes of Haemonchus contortus multidrug-resistant isolated. The GC/MS analysis showed linalool (33.85 %), 1,8-cineole (30.88 %), and δ-terpineol (10.61 %) as the main EO constituents. The LdEO showed an IC50 =0.523 mg/mL in the egg hatch test, and the motility in the adult worm motility test was 95.8 % at 1 mg/mL. The confocal scanning laser microscopy of eggs indicated permeabilization or disruption of egg cell membranes as the possible mechanism of action of LdEO. The scanning electron microscopy of adult worms showed wrinkling, undulations, and cuticular disruptions. The LdEO displayed significant in vitro anthelmintic activity on eggs and adult worms of H. contortus. Additionally, the LdEO showed low oral toxicity in mice at 2,000 mg/kg. Thus, additional in vivo studies are justified to determine its anthelmintic efficacy in small ruminants.

In vitro ovicidal activity of Brongniartia montalvoana against small ruminant gastrointestinal nematodes.

Several plants of the Fabaceae family have been assessed regarding their high nutritional value and anthelmintic properties. The ovicidal effect of the hydroalcoholic extract (Bm-HAE) and subfractions from the aerial parts of Brongniartia montalvoana (Fabaceae) against a mixed strain of gastrointestinal nematodes (GIN) (Haemonchus spp., Trichostrongylus spp. and Oesophagostomum spp.) resistant to albendazole sulfoxide, ivermectin and levamisole was evaluated by the egg hatch test (EHT). The Bm-HAE was subjected to liquid-liquid chemical separation with ethyl acetate giving two fractions, an aqueous (Bm-Aq) and an organic (Bm-EtOAct). The purification of the bioactive fraction (Bm-EtOAct) through chromatographic separation resulted in four bioactive subfractions (BmR6, BmR7, BmR8 and BmR10). The treatments were designed as follows: Bm-HAE at 800, 1,500, 3,000 and 6,000 µg/mL, and Bm-Aq, Bm-EtOAct and subfractions (BmR6, BmR7, BmR8 and BmR10) at 100, 200, 400 and 800 µg/mL. Two properly negative controls (distilled water and 2% methanol) and thiabendazole (100 µg/mL) as a positive control were used for each bioassay. The chemical identification of the extract, fractions and subfractions was performed through chromatographic processes like open column chromatography, thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC-PDA). Additionally, the GIN eggs exposed to the bioactive compounds were observed through confocal laser scanning microscopy (CLSM). The Bm-HAE showed 99.5% egg hatching inhibition (EHI) at 6,000 µg/mL with a lethal concentration (LC50) of 1110 µg/mL. The Bm-EtOAc fraction displayed 99.1% EHI at 800 µg/mL with LC50 = 180 µg/mL. The ovicidal activity of the four subfractions was similar at 800 µg/mL: BmR6 (92% EHI); BmR7 (100% EHI); BmR8 (97.8%); and BmR10 (99.1%). The HPLC-PDA analysis of the bioactive subfractions allowed identification of p-coumaric acid, ferulic acid and coumarin derivatives as major compounds. The CLSM analysis allowed observation of morphological alterations in unhatched larvae caused by bioactive compounds present in the Bm-EtOAc and BmR10. In addition, the flavonoids eriodyctiol, luteolin and cynaroside were described for the first time for B. montalvoana.

In vitro anthelmintic activity of the crude hydroalcoholic extract of Piper cubeba fruits and isolated natural products against gastrointestinal nematodes in sheep.

This study describes the in vitro anthelmintic activity of a hydroalcoholic extract from the fruit of Piper cubeba and its major isolated components against the eggs and larvae of gastrointestinal nematodes obtained from naturally-infected ovines. In vitro anthelmintic activity was evaluated using the egg hatch test (EHT), larval development test (LDT) and L3 migration inhibition test (LMT). The extract showed ovicidal and larvicidal activity, with an EC50 of 200 µg/mL and 83.00 µg/mL in the EHT and LDT, respectively. The extract inhibited 100% of larval migration at the lowest tested concentration (95 µg/mL). The crude extract was purified using successive silica gel chromatographic columns, which revealed the lignans hinokinin, cubebin and dihydrocubebin as the major compounds that were present, which were then used in in vitro tests. Cubebin, dihydrocubebin and hinokinin showed higher activity than the crude extract, with an EC50 for ovicidal activity of 150.00 µg/mL, 186.70 µg/mL and 68.38 µg/mL, respectively. In the LDT, cubebin presented an EC50 of 14.89 µg/mL and dihydrocubebin of 30.75 µg/mL. Hinokinin inhibited 100% the larval development at all concentrations evaluated. In the LMT, dihydrocubebin inhibited 100% the larval migration in all concentrations evaluated while cubebin and hinokinin showed EC50 values of 0.89 µg/mL and 0.34 µg/mL, respectively. P. cubeba extract is rich in several classes of active compounds, but here we demonstrate that the described anthelmintic activity may be related to the presence of these lignans, which are present in larger concentrations than other components of the extract. Our results demonstrate for first time the anthelmintic activity against gastrointestinal nematodes in sheep for this class of special metabolites that are present in P. cubeba fruit. However, future detailed studies are needed to evaluate the effectiveness of P. cubeba fruits extract and active lignans in in vivo tests.

The egg hatch test: A useful tool for albendazole resistance diagnosis in Fasciola hepatica.

In the current study, the egg hatch test (EHT) has been evaluated as an in vitro technique to detect albendazole (ABZ) resistance in Fasciola hepatica. The intra- and inter-assay variations of the EHT were measured by means of the coefficient of variation in different fluke isolates and over time; then, the results of the EHT were compared with the "gold standard" controlled efficacy test, which assesses the in vivo anthelmintic efficacy. The EHT was used later to evaluate the intra-herd variability regarding the level of ABZ resistance in calves infected by the same fluke isolate. Finally, several factors of the initial protocol were modified to improve the simplicity of the assay, including the incubation time of eggs with the drug and the use of eggs collected from faeces. The greatest uniformity between results within the assay and over time until 8 weeks after gallbladder collection (the deadline proposed for egg analysis) was obtained with an ABZ concentration of 0.5 µM. The length of exposure to ABZ was shown to be critical, as prolonged incubation (15 days) led to a change of ovicidal activity. The ABZ concentration of 0.5 µM is suggested as a possible discriminating dose to predict ABZ resistance, due to the close agreement between the results of the EHT at an ABZ concentration of 0.5 µM and those of the in vivo assays.