Ashworthius sidemi Schulz, 1933 and Haemonchus contortus (Rudolphi, 1803) in cervids in France: integrative approach for species identification.

Among gastro-intestinal nematodes, the blood-sucking worms belonging to the subfamily of Haemonchinae are considered to be of pathogenic and economic great importance, particularly in small ruminants. Haemonchus contortus, primary found in domestic ruminants and wild bovines (Mouflon, Chamois), is probably the most studied, but occurrence of Ashworthius sidemi has gradually increased over recent years, especially in Cervids and free roaming wild bovid as the European bison in eastern Europe, and some cases of co-infestation were recently observed on five Roe deer (Capreolus capreolus) and one Red deer (Cervus elaphus) in France. If the diagnosis is possible on the morphological features for adult worms for helminthologists, the identification on some stages (female, subadult, eggs and larvae) is difficult or impossible. Sequencing ND4 domain from the mitochondrial DNA of H. contortus and A. sidemi worms, we observed clearly two distinct clades, with an inter-specific divergence of 28.1%. Basing on this specific domain, a multiplex PCR-based method was developed: new primers were designed and used pooled in one mix PCR, producing amplicons of 454bp for H. contortus and 330bp for A. sidemi, allowing a trivial and an inexpensive taxonomic affiliation after migration. This multiplex PCR-based method was developed here to distinguish H. contortus and A. sidemi regardless their developmental stage, easy to use for highlighting co-infestation cases in both wild and domestic ruminants. It is a non-invasive approach appearing as a good diagnostic tool relevant to coprological cultures.