Pyrantel pamoate

Mice were infected by oral gavage with 200 infective stage 3 (L3) Heligmosomoides polygyrus larvae, diluted in water. The anthelmintic drug pyrantel pamoate (Sigma, 5mg/dose in water) was given orally on two consecutive days. Infections with Plasmodium chabaudi chabaudi (AS) were performed by i.p. injection of 10⁵ parasitized red blood cells. Parasitemia was measured by blinded counting of Giemsa-stained blood smears. Anemia and hemoglobin were measured by diluting blood in Krebs buffered saline with 0.2% glucose and with 100 IU/mL heparin and measured using Vetscan (Abaxis-VetScan HM5 Hematology). Infections with Candida albicans were performed by i.v. injection of 10⁵ yeast forms.

Wild-type female C57BL/6 mice (age 8-10 weeks) were purchased from Janvier Labs (Saint-Berthevin, France). All animals were maintained under specific pathogen-free (SPF) conditions and were fed standard chow ad libidum. H. polygyrus was maintained by serial passage in C57BL/6 mice (H0099/13). Mice were infected with 225 third-stage infective (L3) H. polygyrus larvae via oral gavage in 200µL drinking water. For cure of infection, mice were treated on two consecutive days during the acute stage of infection (day 14-15) with 2mg pyrantel pamoate (Sigma, St. Louis, MO, USA) in 200µL drinking water via oral gavage. On indicated days, mice were sedated via isofluorane inhalation, followed by cervical dislocation. The efficacy of curative treatment with pyrantel pamoate was confirmed at dissection by the lack of adult worms in the small intestine of cured mice. All animal experiments were performed in accordance with the National Animal Protection Guidelines and approved by the German Animal Ethics Committee for the Protection of Animals (LAGeSo, G0176/20).

HD1 Cry5B spore crystal lysate (SCL) and HD1 spore lysate (SL) for in vivo studies were produced and the concentration of Cry5B protein in HD1-Cry5B SCL was determined as previously described [37 (link)]. Tribendimidine was kindly provided by Dr. Shu-Hua Xiao at the Chinese Centers for Disease Control and Prevention. Pyrantel pamoate was purchased from Sigma (Cat# P6210-5G). On the day of use, SL and Cry5B SCL aliquots, tribendimidine powder and Pyrantel pamoate powder were resuspended in distilled water right before in vivo treatment. Cry5B SCL suspension was kept on ice until gavage. When combinations were used, the drugs were given together in a single oral gavage. SL was used as the control for the Cry5B dose-response experiment, but water was used as the control for other experiments. Repeated studies in our lab have shown that SL alone has no effect on parasite burden of fecal egg counts relative to water (manuscript in preparation). Purified Cry5B for cyathostomin developmental inhibition assays was produced as described before [30 (link),38 (link)] and dissolved in 20 mM HEPES (pH8.0) right before setting up the assays.

Wild-type laboratory isolate WMD and multidrug-resistant isolate KGR were described previously (Kitchen et al., 2019 (link)). The KGR isolate used here is resistant to Thiabendazole (TBZ) and IVM. Multidrug-resistant BCR was isolated from a retired racing greyhound, which was raised in Kansas and raced in Florida, in October 2019. The dog had a history of recurring hookworm infections, and had been treated with Drontal Plus (praziquantel/pyrantel pamoate/febantel) prior to adoption. Infective third-stage larvae (iL3s) reared from eggs collected from feces of the infected dog were used to infect a hookworm naïve beagle. All isolates were maintained in beagles as described previously (Schad, 1982 ; Krepp et al., 2011 (link)). Thiabendazole, ivermectin, pyrantel pamoate, dimethyl sulfoxide (DMSO) and 8-bromo-cGMP were purchased from Sigma-Aldrich (St. Louis, MO, USA). Moxidectin (MOX) was a gift from Zoetis (Kalamazoo, MI, USA).