High‐throughput microfluidic real‐time <scp>PCR</scp> as a promising tool in disease ecology

Among recent advances in molecular biology for studying infectious diseases, the microfluidic high-throughput real-time polymerase chain reaction (Htrt PCR) has emerged as an efficient first-line tool for the detection of a wide range of infectious agents (IA) in a host system. This technology allows large batches of samples to be screened simultaneously for tens of targeted IA by real-time PCR. It represents a promising tool in disease ecology. As a proof of concept, we present here the development steps and initial application of a microfluidic Htrt PCR system for the detection of DNA from 28 selected IA in a set of wild vertebrates. We applied this approach to 497 samples (mainly mucosal swabs and necropsy tissues) from 274 seabirds and 80 mammals from Southern Ocean islands. This ecosystem is of particular interest for disease ecology and biodiversity conservation due to the high host densities of breeding colonies, within and among which pathogens may spread rapidly. Positive samples were subsequently confirmed for each PCR system using a secondary real-time or conventional PCR system and/or sequencing. Fourteen targeted IA were detected. The approach allowed an efficient screening of host species for known seabird pathogens, including Pasteurella multocida (avian cholera) (9.9% [6.6-14.0] in seabirds, 18.3% [9.5-30.4] in mammals) and Erysipelothrix amsterdamensis (15% [11-19.8] in seabirds, 2.1% [0.4-6.1] in mammals) in cloacal and rectal swabs. Their detection on islands where they were not known previously raised conservation concerns. IA not previously known to be circulating in the system were detected at high prevalence, notably Chlamydiaceae on all colonies (32.1% [26.6-38] in seabirds and 21.3% [12.9-31.8] in mammals, in cloacal and rectal swabs). Prevalence and diversity of targeted IA could be compared among sites and host species, highlighting the usefulness of the approach to explore drivers of IA community dynamics, but also to identify specific hosts as potential epidemiological sentinels or reservoirs. Htrt PCR is a powerful and versatile tool that can be used in disease ecology for exploring the composition of IA communities within host communities, but also for addressing other important basic and applied questions in multi-host, multi-pathogen systems.