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BackgroundAnthelmintic 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.MethodsThree 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.ResultsA 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.ConclusionsOverall, 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.

The aim of this study was to investigate genetic selection and P-glycoprotein (PGP) expression in three different isolates of Cooperia oncophora before treatment and after ivermectin (IVM) injection. Adult parasites were recovered from nine calves experimentally infected with the isolates represented by one IVM susceptible laboratory isolate, and two field isolates showing signs of phenotypic macrocyclic lactone resilience according to the faecal egg count reduction test. Five males and five females per isolate were examined both pre- and post-IVM treatment giving a total of 60 worms. A sequence from C. oncophora (Con-pgp) was identified, showing 83 % similarity to Pgp-9 of Caenorhabditis elegans. Primers specific to putative Con-pgp-9 mRNA were designed, generating a 153-bp PCR product. Total RNA was prepared from all worms, and Con-pgp-9 expression was measured by quantitative real-time reverse transcription PCR. Our results showed that mean PGP concentrations were four to five times higher in female as compared to male worms. No significant differences in gene expression between experimental groups pre- and post-IVM selection were detected. However, PGP gene expression tended to be increased by IVM treatment in male worms (p = 0.091), with 70 % higher mean expression in treated than in untreated male worms. Amplified fragment length polymorphism analysis did not demonstrate any bottleneck effect within the different isolates post-treatment. The total mean gene diversity values were 0.158 and 0.153 before and after treatment, respectively. Inbreeding coefficient in subpopulations compared to total population FST was 0.0112, suggesting no genetic differentiation between or within the investigated isolates in relation to treatment. In conclusion, comparison of Con-pgp-9 expression showed no significant difference before and after treatment, but some tendency towards increasing expression in male worms.

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