Biology and distribution of red mark syndrome associated Midichloria-like organism in a rainbow trout farm.

Red Mark Syndrome (RMS) is a disease affecting rainbow trout (Oncorhynchus mykiss), characterized by distinct red skin lesions, particularly in market-size individuals. While RMS is non-lethal, it leads to significant economic losses associated with product downgrading. Increasing evidence suggests the involvement of an uncultured bacterium belonging to the Midichloriaceae family (order Rickettsiales), hereafter referred to as the RMS-Midichloria-like organism (RMS-MLO), as causative agent of the disease. This study aims to shed light on the biology of RMS-MLO and its potential role in RMS pathogenesis, using a multifaceted investigative approach. The study was conducted at a single trout farm, sampled monthly over the course of a year, representing the first investigation to monitor the system beyond periods of active disease outbreaks. Environmental DNA (eDNA) was extracted from water and sediment samples collected from fish tanks and analyzed via qPCR to detect the presence of RMS-MLO. These eDNA samples were also screened for Ichthyophthirius multifiliis, a ciliate parasite hypothesized to act as a vector for RMS-MLO. Fish were sampled both during outbreak periods and in their absence, and subjected to bacteriological, parasitological, and histological analyses. Tissue samples from affected skin lesions were processed for transmission electron microscopy (TEM) to localize the bacterium at the tissue and subcellular level. To identify other potential vectors or reservoirs among the tank-resident micro- and meiofauna, individual organisms were isolated from the water, propagated in the lab when feasible, and tested by qPCR for RMS-MLO DNA. Additionally, eukaryotic metabarcoding of eDNA is currently being conducted to explore correlations between community composition and RMS-MLO occurrence. Molecular analyses revealed that RMS-MLO was present in some fish tanks throughout the year, with up to 89% of water and sediment samples testing positive at specific time points. Typical RMS lesions were observed in fish sampled in March and April. The bacterium was identified in tissue sections, particularly within muscle cells. DNA of I. multifiliis was detected in a subset of water and sediment samples, with a maximum occurrence of 45% at certain time points. Furthermore, RMS-MLO DNA was detected in association with various protists (e.g., amoebae, ciliates) and metazoans (e.g., nematodes, arthropods, annelids), suggesting that multiple organisms in the aquatic environment may be involved in the transmission or persistence of the bacterium.