Faecal egg count reduction test, deep amplicon sequencing of isotype-1 β-tubulin gene and in ovo larval development assay reveal susceptibility to benzimidazoles of porcine nematodes Oesophagostomum spp. and Ascaris suum in outdoor-reared pigs in Germany

Abstract

Oesophagostomum spp. and Ascaris suum represent the most common porcine nematodes and anthelmintic resistance (AR) to various anthelmintics has been reported for Oesophagostomum. However, the current AR status for worm populations on German farms and practical methods facilitating reliable AR detection are missing. Herein, the efficacy of benzimidazoles (BZ) (fenbendazole, 5 mg/kg body weight, single dose) was analysed on 13 farms with outdoor access. The Faecal Egg Count Reduction Test (FECRT) estimates for strongyles on the farms (range 99.8–100 %) exceeded the target efficacy (99 %) of the new W.A.A.V.P. guideline for Oesophagostomum dentatum. Deep amplicon sequencing was used for the first time for porcine nematodes and revealed no polymorphisms associated with BZ-resistance in codons 134, 167, 198 and 200 of the isotype-1 β-tubulin gene. Nemabiome analysis using ITS-2 deep amplicon sequencing, based on two pre- and post-treatment samples, showed a significant increase (p < 0.001) of Oesophagostomum quadrispinulatum after BZ treatment. For A. suum, the interpretation of FECRT estimates can be hindered due to coprophagy-associated false-positive egg counts in pigs. Therefore, two FECRT analysis for A. suum were pursued, the first analyses included all EPG data, the second considered EPGs <200 pre- and post-treatment as negative. An in ovo larval development assay (LDA) was developed for the in vitro analysis of BZ-susceptibility in A. suum. Computed EC50 values ranged from 1.50 to 3.36 μM thiabendazole (mean 2.24 μM). An EC50 of 3.90 μM thiabendazole (mean EC50 + 3 × SD) as provisional cut-off for detection of resistant populations is suggested. In conclusion, no AR was detected in Oesophagostomum using the FECRT and β-tubulin deep amplicon sequencing. For A. suum the FECRT results were ambiguous, in some cases even when excluding the low egg counts from calculations. With the in ovo LDA all investigated A. suum populations were identified as susceptible to BZ.