Phytochemical modulation of P-Glycoprotein and its gene expression in an ivermectin-resistant Haemonchus contortus isolate in vitro.

Haemonchus contortus is the most important gastrointestinal nematode in small ruminant systems worldwide and has developed resistance to several drugs, including ivermectin (IVM). IVM is not only a veterinary drug but also a safe, broad-spectrum, antiparasitic drug used in humans. One of the main IVM-resistance mechanisms in H. contortus involves P-glycoprotein (PgP), a trans-membrane transport protein that rids worm cells from toxic molecules. This study aimed to evaluate the anthelmintic activity of IVM, alone or combined with main terpenes of essential oils (alpha-terpinene, beta-citronellol, beta-pinene, citronellal, limonene, menthol, and terpinolene) and with phenolic compounds (epicatechin, epigallocatechin, gallocatechin, pentagalloylglucose, procyanidin, and quercetin). All compounds were tested, alone or combined with IVM, against susceptible (HcS) and resistant (HcR) isolates of H. contortus through the larval development test (LDT) and the adult motility assay (AMT) using verapamil (VP), a known PgP modulator, as a control. Results for the LDT determined that the lethal concentration required to kill 50% of nematodes (LC50) with IVM was 10 times greater (0.01 µg/mL) for HcR than for HcS (0.001 µg/mL). The combination IVM + VP inhibited the activity of PgP in HcR resulting in a LC50 = 0.002 ug.mL-1. Although limonene was the least effective and alpha-terpinene the most effective terpene when tested alone against HcR, the best combinations were IVM + limonene and IVM + quercetin both produced LC50 = 0.002 µg/mL (similar to IVM+VP) which were chosen for subsequent tests. Because adult parasites are the final target for anthelmintics, IVM was evaluated in HcS (LC50 = 0.067 µg/mL) and HcR (LC50 =164.94 µg/mL) through the AMT. Results obtained with IVM + VP (LC50 = 0.020 µg/mL) in HcR were similar to IVM + limonene (LC50 = 0.028 µg/mL) and outperformed IVM + quercetin (LC50 = 1.39 µg/mL). RNA extracts from HcR adult worms exposed to IVM, IVM+VP, and IVM + limonene were evaluated for PgP expression by RT-PCR. For most concentrations, PgP-9 was significantly more expressed in worms treated with IVM alone than in worms treated with IVM + VP or IVM + limonene. Our results suggest that limonene is involved in the modulation of the PgP-9 gene and that it can restore the activity of IVM in the HcR isolate down to levels seen in HcS. Limonene is one of the main compounds found in citrus peel and has the potential to be both safe and affordable if used in combination with IVM to restore its anthelmintic effects against multi-drug-resistant H. contortus isolates. Our results also suggest that we may be more successful by combining natural products with failing commercial anthelmintics than trying to find natural substitutes for them.

Evaluation of Parasitological Homeopathic Complex in the Control of Gastrointestinal Nematodes in Peripartum Sheep.

BACKGROUND: The appearance of anthelmintic resistance has made it difficult to control verminosis in sheep, leading to increased research to find alternative nematode control. The use of homeopathy in veterinary medicine has been studied as an alternative for the treatment and control of some diseases. In this study, ewes received an anti-parasitic homeopathic complex medicine during the critical peripartum period of increased susceptibility to nematodes. METHODS: Three randomized groups containing 16 animals each were assigned as follows: 'H10' received 10 g homeopathic complex added to concentrated food per day; 'H20' received 20 g homeopathic complex added to concentrated food per day; 'C' (control group) did not receive a homeopathic complex. Animals were tested to evaluate the effect of homeopathy on several health parameters during a period of 110 days. RESULTS: The parasite that prevailed in the copro-cultures of both treatments throughout the experiment was Haemonchus contortus (78.26%). Packed cell volume averages did not present statistically significant differences between the treatments (24.5, 24.4 and 23.9% to C, H10 and H20, respectively; p < 0.05). For total white cell count, lower mean values ( ± standard error of mean) were observed for the H20 treatment (5,490.9 ± 0.02/µL; p < 0.05), whereas the H10 (5,919.4 ± 0.02/µL) and control (6,098.5 ± 0.02/µL) presented higher and similar averages (p > 0.05). The values for erythrocytes, monocytes, and lymphocytes did not show differences between treatments (p > 0.05). Body weight was greater in the H10-treated animals compared with control. For the fecal egg count (FEC) of Trichostrongylidae and Strongyloides spp., respectively, the averages of the H20 treatment (1,523.0 and 30.6) were not different from control (1,616.0 and 31.6) and H10 (1,038.0 and 27.6); for Trichostrongylidae, however, H10 presented a lower FEC than the control (p = 0.02). For Cooperia, H10- and H20-treated animals showed FEC reductions of 97% and 98%, respectively. CONCLUSIONS: H20 treatment in peripartum sheep resulted in greater body weight and lower leukocyte count. H10 was associated with lower FEC for Trichostrongylidae. Both H10 and H20 justify a label of 'effective anthelmintic' for Cooperia.