Development of In Vitro Assays with the Canine Hookworm Uncinaria stenocephala and Assessment of Natural Plant Products for Anti-Parasitic Activity

Abstract

Enteric helminth infection is an increasing concern in companion animals due to reports of resistance to commonly used anthelmintic drugs. Thus, the assessment of new therapeutic options such as bioactive dietary additives is of high importance. Here, we adapted egg hatch, larval migration, and larval motility assays to screen extracts of several natural ingredients against the canine hookworm Uncinaria stenocephala, a prevalent parasite of dogs in northern Europe. Egg hatch and larval migration assays were established showing that the anthelmintic drugs levamisole and albendazole had strong anti-parasitic activity against U. stenocephala, validating the use of these assays for the assessment of novel anti-parasitic substances. Subsequently, we identified that extracts from the seaweed Saccharina latissima, but not extracts from grape seed or chicory, significantly inhibited both hatching and larval migration. Finally, we showed that α-linolenic acid, a putative anti-parasitic compound from S. latissima, also exhibited anti-parasitic activity. Collectively, our results established a platform for the screening for anthelmintic resistance or novel drug candidates against U. stenocephala and highlighted the potential use of seaweed extracts as a functional food component to help control hookworm infection in dogs.

Keywords: Uncinaria stenocephala; anthelmintic; hookworms; in vitro; seaweed.

Figure 1

Egg hatch assays with Uncinaria stenocephala eggs—eggs were incubated with either water, 1% DMSO, albendazole (ABZ) or levamisole (LEV)—both 50 µg/mL, trans-cinnamaldehyde (Cinn; 10 µg/mL), chicory extract (Chic; 1 mg/mL), grape seed extract (Grape; 1 mg/mL), or 4 different Saccharina latissima extracts (SW1-SW4; see Materials and Methods for description; 1 mg/mL). Hatched eggs were counted after 48 h. Data are presented as mean ± S.E.M. (n = 6 for each treatment). ** p < 0.01 by ANOVA.

Figure 2

Larval migration assays with Uncinaria stenocephala larvae—larvae were incubated with culture media together with either water, 1% DMSO, levamisole (50 µg/mL), trans-cinnamaldehyde (Cinn; 10 µg/mL), chicory extract (Chic; 1 mg/mL), Grape seed extract (Grape; 1 mg/mL), or 4 different Saccharina latissima extracts (SW1-SW4; see Materials and Methods for description; 1 mg/mL). Migrated larvae were counted after 24 h incubation. Data are presented as mean ± S.E.M. (n = 9 for each treatment). * p < 0.05; ** p < 0.01 by ANOVA.

Figure 3

Larval migration assays with different doses of Saccharina latissima extract—Uncinaria stenocephala third stage larvae were incubated with different doses of a dichrolomethane-methanol extract of S. latissima sourced from Grenå, Denmark. Migrated larvae were counted after 24 h incubation. Data are presented as mean ± S.E.M. (n = 3 for each treatment).

Figure 4

Larval migration assays with different doses of α-linolenic acid—Uncinaria stenocephala third stage larvae were incubated with different doses of α-linolenic acid. Migrated larvae were counted after 24 h incubation. Data are presented as mean ± S.E.M. (n = 4 for each treatment).