Macrocyclic lactone anthelmintic-induced leukocyte binding to Dirofilaria immitis microfilariae: Influence of the drug resistance status of the parasite

Abstract

The macrocyclic lactone anthelmintics are the only class of drug currently used to prevent heartworm disease. Their extremely high potency in vivo is not mirrored by their activity against Dirofilaria immitis larvae in vitro, leading to suggestions that they may require host immune functions to kill the parasites. We have previously shown that ivermectin stimulates the binding of canine peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes (PMNs) to D. immitis microfilariae (Mf). We have now extended these studies to moxidectin and examined the ability of both drugs to stimulate canine PBMC and PMN attachment to Mf from multiple strains of D. immitis, including two that are proven to be resistant to ivermectin in vivo. Both ivermectin and moxidectin significantly increased the percentage of drug-susceptible parasites with cells attached at very low concentrations (<10 nM), but much higher concentrations of ivermectin (>100 nM) were required to increase the percentage of the two resistant strains, Yazoo-2013 and Metairie-2014, with cells attached. Moxidectin increased the percentage of the two resistant strains with cells attached at lower concentrations (<10 nM) than did ivermectin. The attachment of the PBMCs and PMNs did not result in any parasite killing in vitro. These data support the biological relevance of the drug-stimulated attachment of canine leukocytes to D. immitis Mf and suggest that this phenomenon is related to the drug resistance status of the parasites.

Keywords: Dirofilaria immitis; Drug resistance; Ivermectin; Moxidectin; Peripheral blood mononuclear cells; Polymorphonuclear leukocytes.

Graphical abstract

Fig. 1

Effect of ivermectin and moxidectin on PMN and PBMC attachment to microfilariae of the Missouri and Georgia-2 strains. A) Effect of ivermectin on attachment of PMNs. B) Effect of moxidectin on attachment of PMNs. C) Effect of ivermectin on attachment of PBMCs. D) Effect of moxidectin on attachment of PBMCs. For each panel the bars represent the average percentage of Mf with at least one cell attached at varying concentrations of the drug; from left to right these were 0, 1, 3, 10, 30, 100, 300 and 1000 nM. MO = Missouri, GA-2 = Georgia-2. In each case N = 4, each experiment carried out in triplicate. Error bars indicate the standard error of the mean.

Fig. 2

Effect of ivermectin and moxidectin on PMN and PBMC attachment to microfilariae of D. immitis strains with suspected resistance against at least one macrocyclic lactone. A) Effect of ivermectin on attachment of PMNs. B) Effect of moxidectin on attachment of PMNs. C) Effect of ivermectin on attachment of PBMCs. D) Effect of moxidectin on attachment of PBMCs. For each panel the bars represent the average percentage of Mf with at least one cell attached at varying concentrations of the drug; from left to right these were 0, 1, 3, 10, 30, 100, 300 and 1000 nM. YAZ = Yazoo-2013, MET = Metairie-2014, JYD = JYD-27. In each case N = 4-5, each experiment carried out in triplicate. Error bars indicate the standard error of the mean.

Fig. 3

Survival of Missouri Mf in the presence of canine PBMCs and varying concentrations of ivermectin. Mf were cultured together with PBMCs for 5 days in different concentrations of ivermectin, and the number of motile larvae counted as a proportion of those originally added. N = 3. Error bars indicate the standard error of the mean.