A controlled study on gastrointestinal nematodes from two Swedish cattle farms showing field evidence of ivermectin resistance

Abstract

Background: Anthelmintic resistance (AR) is an increasing problem for the ruminant livestock sector worldwide. However, the extent of the problem is still relatively unknown, especially for parasitic nematodes of cattle. The effect of ivermectin (IVM) (Ivomec inj.®, Merial) was investigated in Swedish isolates of gastrointestinal nematode (GIN) populations showing signs of AR in the field to further characterise the AR status by a range of in vivo and in vitro methods.

Methods: Three groups, each of 11 calves, were infected with an equal mixture of third stage larvae (L3) of Cooperia oncophora and Ostertagia ostertagi. Group A was inoculated with an IVM-susceptible laboratory isolate and groups B and C with isolates originating from 'resistant' cattle farms. Faecal egg counts (FEC) were monitored from 0 to 45 days post infection (d.p.i.), and L3 were harvested continuously for larval migration inhibition testing (LMIT) and species-specific PCR (ITS2). At 31 d.p.i., one calf from each group was necropsied and adult worms were recovered pre-treatment. At 35 d.p.i., calves from all groups were injected with IVM at the recommended dose (0.2 mg/kg bodyweight). At 45 d.p.i., another two animals from each group were sacrificed and established gastrointestinal worms were collected and counted.

Results: A few animals in all three groups were still excreting eggs (50-150 per g faeces) 10 days post IVM injection. However, there was no significant difference in the FEC reductions in groups A (95%; 95% CI 81-99), B (98%; 92-100) and C (99%; 97-100) between 35 and 44 d.p.i. Furthermore, LMIT showed no significant difference between the three groups. Approximately 100 adult O. ostertagi were found in the abomasum of one calf (group B), whereas low to moderate numbers (400-12 200) of C. oncophora remained in the small intestine of the calves in all three groups at 45 d.p.i. PCR on L3 harvested from faecal samples up to 10 days post treatment showed a ratio of 100% C. oncophora in the calves inoculated with isolates A and B, whereas C also had 8% O. ostertagi.

Conclusions: Overall, this experiment showed that the animals were successfully treated according to the Faecal egg count reduction test (FECRT) standard (≥ 95% reduction). However, several adult worms of the dose-limiting species C. oncophora demonstrably survived the IVM treatment.

Figure 1

Faecal egg count patterns. Mean faecal egg count patterns over time (McMaster method), showing a significant difference (p = 0.002) in egg shedding patterns between isolates (groups) A, B and C from 1-45 d.p.i. However, there was no significant difference (p = 0.34) when comparing the egg shedding levels of the three groups before treatment, 1-35 d.p.i. Group A was infected with a propagated IVM-susceptible laboratory isolate and groups B and C with two different Swedish field isolates showing phenotypic clinical IVM resistance in field trials. All calves were treated with injectable IVM at 35 d.p.i.

Figure 2

Mean egg output reduction. Mean egg output reduction (95%CI) measured by (a) the McMaster method and (b) the FLOTAC method in isolates 9 days after treatment (44 d.p.i.) with IVM. Calves in group A were infected with a propagated IVM-susceptible laboratory isolate and those in groups B and C with two different field isolates showing phenotypic clinical IVM resistance in field trials. Reductions were similar in all three groups.

Figure 3

Adult C. oncophora recovered at necropsy post treatment. Boxplot of estimated numbers of adult C. oncophora worms recovered at necropsy of calves 10 days after injection (45 d.p.i.) with IVM. Calves were previously infected with a mixture of C. oncophora and O. ostertagi, representing isolates with different deworming histories. Isolate A was propagated IVM-susceptible, whereas B and C were two different field isolates showing phenotypic clinical IVM resistance in field trials. Boxplots show larger variation within groups than between groups.

Figure 4

Dose-response curves, larval migration inhibition test. Dose-response curves of the data obtained in LMIT with the three different isolates of L3 larvae, showing no significant difference between groups. Calves in group A were infected with equal mixtures of laboratory-maintained C. oncophora and O. ostertagi from TiHo, and those in groups B and C with cattle nematodes from two different farms in Uppland, Sweden, showing phenotypic clinical IVM resistance in previous field trials.