Copper chloride and copper sulphate in combination with nitroxynil against gastrointestinal nematodes of ruminants: A possible hitchhiking synergic effect at low concentrations.

Infections caused by Haemonchus spp. and Trichostrongylus spp. are major health problems for sheep and cattle. The objective of this study was to determine the efficacy of copper chloride (CuCl2), and copper sulphate (CuSO4) at 2.0, 7.0, 30.0, 125.0, 500.0, and 2000.0 µM formulations, and nitroxynil 34% (NTX) at 0.235 mM against gastrointestinal nematodes (GINs) of ruminants. Hence, the in vitro egg hatch test (EHT), the larval development test (LDT), and the larval migration inhibition test (LMIT) were used. Haemonchus spp. (52%) and Trichostrongylus spp. (38%) were the most frequently found parasites. The data fitted a concentration-dependent shape with the highest efficacies of CuCl2 and CuSO4 at 95.2 and 97.3% for parasites collected from sheep, and 95.8 and 93.4% from cattle, respectively. The combination of the 50% inhibitory concentration (IC50) of CuCl2 and CuSO4 and the IC10 of NTX showed up to a 52% increase in efficacy above the expected additive results, demonstrating a synergic/drug enhancer interaction. NTX may retain Cu-II ions by complexation, in a hitchhiking mechanism carrying the salts across the parasite cell wall, causing oxidative stress as a consequence of free radical production and cell damage. Synergy data between NTX and CuCl2, and CuSO4 represent a viable opportunity to develop new formulations for combating parasites of ruminants (i.e., Fasciola hepatica, Haemonchus spp., and Oesophagostomum spp.).

Mentha villosa Hubs., M. x piperita and their bioactives against gastrointestinal nematodes of ruminants and the potential as drug enhancers.

Parasitic diseases caused by gastrointestinal nematodes (GIN) are responsible for a major impact on ruminant welfare. Although the available anthelmintics have a safe margin of toxicity to the animals, their indiscriminate use has increased the selection of resistant parasite populations. In this scenario, essential oils (EO) stand out as a promising ecofriendly therapeutic alternative against GIN. The objective of this work was to determine the effect of the EO of Mentha villosa Hubs (MVEO) collected in 2017 and 2018, M. x piperita (MPEO) and their main components, carvone and limonene, against the third stage larvae (L3) of Haemonchus spp. and Trichostrongylus spp. The solutions, including in nanoemulsion preparations, were tested in a range of concentrations using the larval migration inhibition test (LMIT). The EO and carvone were also tested in combination with nitroxynil (NTX) to determine their effect as drug enhancers (additive or synergy). MVEO/2017, MVEO/2018, MPEO and carvone showed 70.6 (73.4 mg/mL), 86.3 (74.9 mL/mL), 95.5 (143.6 mg/mL), and 88.2 % (38.3 mg/mL) efficacy against L3, respectively. Carvone alone had approximately a 3-fold higher efficacy when compared to its concentration in each EO: 68.8 % in MVEO/2017 and 83.9 % in MVEO/2018. Limonene did not show any significant effect on inhibiting L3 migration. The combination of MPEO and NTX, and carvone and NTX showed a statistically significantly (P <  0.05) synergic and additive effect, respectively, when compared to the isolated treatment. The nanoemulsion of MVEO/2017 at 0.367 mg/mL, inhibited L3 migration by 83.1 %, demonstrating to be highly effective (concentration ratio of 1:0.004), when compared to the MVEO/2017 (70.6 % at 73.4 mg/mL) extraction. The in vitro data from the combination of MPEO or carvone plus NTX suggest that these products can be considered for in vivo experiments against the most important GIN of ruminants as drug enhancers, possibly reducing the final concentration of NTX`. The efficacy of carvone was higher (EC50 = 1.96 mg/mL) than its expected efficacy, based on its concentrations on both EO. Therefore, this component does not need the entire EO composition to exert its L3 motility action. The remarkable efficacy demonstrated by the MVEO/2017/nanoemulsion (EC50 = 0.10 mg/mL), supports its potential to be a candidate to the next-generation therapy to alleviate clinical parasite infections and combat GIN resistant populations.