Haemonchus contortus strains
Two isolates of H. contortus were used in the study:
i) Haecon-5 strain: Presented by Professor Robin B. Gasser of the University of Melbourne; susceptible to all commercial anthelmintics.
ii) Haemonchus contortus Zhaosu strain: collected from the abomasum of dead sheep in Zhaosu County, Xinjiang Uygur Autonomous Region, China in 2017 (43°09′～43°15′N 80°08′～81°30′E). This region has an average annual rainfall of 511.8 mm, with long cool winter and short mild summer (Fig. 1).
The commercial IVM powder (Shyuanye Inc, Shanghai, China) was used as a source of IVM in LDA. The stock solution (1 mg/mL) was prepared in dimethyl sulfoxide (DMSO) and was diluted in DMSO to generate a series of working solutions. IVM Injection (Yuanzheng Pharmaceutical Co., Hebei, China) was used in the faecal egg count reduction test (FECRT).
Culture of larvae and morphological larval identification
The H. contortus female adults collected from the abomasum of dead sheep from Zhaosu were minced and then mixed with the sterilized sheep feces, which were then submitted to the College of Veterinary Medicine, Huazhong Agricultural University, Wuhan. The sample was incubated at 27 °C for 7 days, subsequently the third-stage larvae (L3) of Zhaosu strain of H. contortus were collected and morphologically identified. In order to further perform FECRT, around 6-months old goats free of parasites were infected with 8000 L3s per goat of Zhaosu strain and Haecon-5 strain, respectively. All experimental goats used in this study were housed in a cage without access to grass and feces.
Recovery of H. contortus eggs
Fresh H. contortus eggs were recovered from the faeces of each donor goat using a similar method from a previous report . In short, approximately 100 g of faeces were macerated in a mortar with 800 ml of sugar water. The suspension was filtered using the 450 μm and 125 μm fine sieves and filtrate was covered with a piece of plastic film by setting on the surface of suspension liquid for about 40 minutes. Then, the plastic film was removed and its surface was washed with normal saline. The collection solution was further filtered using the 50 μm and 38.5 μm fine sieves, the eggs on the surface of the sieves (38.5 μm) are collected and used immediately for larval development assays.
Larval development assay (LDA)
Laval development assay (LDA) was carried out as modified from the original method . Briefly, the egg suspension (2 ml) recovered from faeces was added to T25 cell bottle containing 400 μl of growth medium (1 g yeast extract plus 90 ml dH2O; autoclaved for 15 min; added with 10 ml of 10× concentrated Earles’s solution). Each bottle contains about 5000 eggs which were incubated at 27 °C for 24 h. On the next day, after the eggs hatched, 99 μl of larval suspension (~100 larvae) and 1 μl IVM working solution (final DMSO concentration in experimental group and control wells was 1% v/v with triplicate assay wells) were added to the wells of 96-well plates and incubated at 27 °C for another 6 d (Fig. 2A). Final IVM concentrations ranged from 12.5 ng/μl to 0.2 ng/μl (12.5, 10.0, 5.0, 2.5, 1.25, 0.4, 0.2) for Zhaosu strain and 5 ng/μl to 0.1 ng/μl (5.0, 2.5, 1.25, 0.41, 0.205, 0.1 ng/μl) for Haecon-5 strain, control group received DMSO alone. At the end of the test, the number of L3s was counted in each well and the resistance ratio (RR) was calculated .
Faecal egg reduction test
A faecal egg count reduction test (FECRT) was performed according to the World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines for evaluation of anthelmintic efficacy in ruminants . In an attempt to further confirm the results of the LDA and the cause of death of sheep, four consecutive efficacy trials involving H. contortus experimental infections were carried out in goat. Thirty days after infection, goats in the treatment group were treated with IVM (IVM sub-cutaneous injection) at different doses including 0.2 mg/kg (standard dose), 0.4 mg/kg (2 times dose), 0.6 mg/kg (3 times dose) and 0.96 mg/kg (4.8 times dose), respectively. Goats in the control group were left untreated. One day before the treatment and 14 d after the treatment, goats were checked for faecal egg counts (EPG) and the efficacy of the treatment was evaluated by the faecal egg count reduction test (FECRT), according to the formula, based on the guidelines recommended by WAAVP . In addition, after 20 days of treatment at 0.4 mg/kg, the goats infected with Zhaosu strain were slaughtered to collect adults and calculate the worm burden. Percentage faecal egg count reduction (FECR) was determined according to the formula FECR (%) =（1- [T2/TI ]）×100 where T1 (before treatment) and T2 (post treatment) are the arithmetic mean egg counts in the group treated with IVM, respectively .