A novel duplex ddPCR assay for detection and differential diagnosis of Ascaridia galli and Heterakis gallinarum eggs from chickens feces

Since the EU ban on battery cages, many studies have listed Ascaridia galli and Heterakis gallinarum as most common roundworms in European laying hen population. A complicating factor is that eggs of these parasites are almost identical. Thus, lack molecular diagnostic approaches has driven epidemiological to take necropsy for species discrimination, which labor cost intensive. Here, we describe a novel tool based droplet digital PCR simultaneous identification absolute quantification both ascarids chickens’ droppings using two different genus-specific primer-probe sets targeting second internal transcribed spacer region (ITS-2) nuclear ribosomal (rRNA) gene array. No cross-reaction was observed when combinations DNA species-specific primers probes were tested. The lowest obtained frequency threshold detection H. presence constant A. concentration determined be 0.8 %. After validation, used assay analyze field samples collected from several Swedish farms. Out 134 samples, 86 (64 %) positive while 11 (8.3 gallinarum. These initially analyzed with flotation technique ascarid eggs. results Cohen’s kappa indicated substantial agreement (85.8 between tests. In conclusion, validated molecular-based differentiation intestinal major importance chickens high precision. Although this study focuses hens, findings may well bearing all types chicken production systems. present lays groundwork future research into epidemiology important parasite species.