Red mark syndrome in rainbow trout (Oncorhynchus mykiss): etiological and diagnostic investigations

The subject of this PhD project has been a non-lethal skin disease affecting farmed rainbow trout worldwide and named red mark syndrome (RMS). To date, the etiology of this syndrome is still unknown, but a Midichloria-like organism (MLO) is strongly believed to be associated with it. As reported by the results of the 25th Annual Workshop of the European Reference Laboratories for Fish Diseases (DTU-AQUA, Denmark), RMS remains to date a major concern in rainbow trout farming. This disease does not cause relevant mortality nor has any impact on growth rate, but it does cause a marked downgrade of commercial products. The objectives of the research studies described here are to contribute to create a gold standards diagnostic approach, to explore novel diagnotic techniques, especially rapid methods for early detection, and to contribute to the definition of a causative agent for RMS. Most importantly the work developed during this PhD aimed at establishing a network of collaborative institutions and companies, both on a national and international level. Each chapter of this thesis represents the description of one of these objectives. The first phase of this project consisted of a trial, which tested the effect of 3 temperatures on the transfer of MLO from RMS-affected fish to naïve SPF cohabitants at the DTU-AQUA (Denmark) facilities. Through electron microscopy, this study visualized for the first time MLOs in fish affected by RMS in experimental conditions. Furthermore, a strong association between the disease and the presence of MLO was established. This research project gained funds and support of Aquaexcel 2020, European Association of Fish Pathologists (EAFP), and MSD Animal Health UK. Skin and spleen samples taken from the experimental infection were then used both to create a histological scoring system, and to develop and apply a sensitive droplet digital PCR for the detection of MLO. The histological scoring system was modified starting from the results of a dedicated study in the field, including healing stages as well. The development and application of a specific ddPCR technique, was possible through the cooperation between DI4A and human clinical pathology laboratory at the human hospital of Udine. This extremely sensitive molecular technique was then used to detect and quantify MLO DNA from different environmental sources like water, blood and sediment both in field and experimental cases. Through the course of the first and second year of the PhD, the collaboration with Moredun institute of research (Scotland) and Aquatic Vets Ltd. made possible the publication of the first critical review article on RMS disease. Experience from field cases lead to the first signalament of the disease in Bosnia-Herzegovina, whereas during 2022 an outbreak of RMS was reported in a RAS system in Slovenia. For the first time RMS was described in juvenile rainbow trout as small as 35grs and MLO DNA was extracted from water samples. During the course of the project, we collected epidemiological data from more than 100 Italian rainbow trout farms through ad hoc questionnaire and map the presence of RMS across Italy. The present work could represent the foundation for future studies focused towards the complete molecular characterisation of MLO. Together with this, the use of ddPCR applied to MLO DNA and environmental DNA can certainly help to implement both biosecurity measures and lead to early diagnosis or even prediction of RMS outbreaks. Finally, studies of economic impact of this disease are needed to develop possible management strategies and options, especially from a commercial point of view.

The subject of this PhD project has been a non-lethal skin disease affecting farmed rainbow trout worldwide and named red mark syndrome (RMS). To date, the etiology of this syndrome is still unknown, but a Midichloria-like organism (MLO) is strongly believed to be associated with it. As reported by the results of the 25th Annual Workshop of the European Reference Laboratories for Fish Diseases (DTU-AQUA, Denmark), RMS remains to date a major concern in rainbow trout farming. This disease does not cause relevant mortality nor has any impact on growth rate, but it does cause a marked downgrade of commercial products. The objectives of the research studies described here are to contribute to create a gold standards diagnostic approach, to explore novel diagnotics techniques, especially rapid methods for early detection, and to contribute to the definition of a causative agent for RMS. Most importantly the work developed during this PhD aimed at establishing a network of collaborative institutions and companies, both on a national and international level. Each chapter of this thesis represents the description of one of these objectives. The first phase of this project consisted of a trial, which tested the effect of 3 temperatures on the transfer of MLO from RMS-affected fish to naïve SPF cohabitants at the DTU-AQUA (Denmark) facilities. Through electron microscopy, this study visualized for the first time MLOs in fish affected by RMS in experimental conditions. Furthermore, a strong association between the disease and the presence of MLO was established. This research project gained funds and support of Aquaexcel 2020, European Association of Fish Pathologists (EAFP), and MSD Animal Health UK. Skin and spleen samples taken from the experimental infection were then used both to create a histological scoring system, and to develop and apply a sensitive droplet digital PCR for the detection of MLO. The histological scoring system was modified starting from the results of a dedicated study in the field, including healing stages as well. The development and application of a specific ddPCR technique, was possible through the cooperation between DI4A and human clinical pathology laboratory at the human hospital of Udine. This extremely sensitive molecular technique was then used to detect and quantify MLO DNA from different environmental sources like water, blood and sediment both in field and experimental cases. Through the course of the first and second year of the PhD, the collaboration with Moredun institute of research (Scotland) and Aquatic Vets Ltd. made possible the publication of the first critical review article on RMS disease. Experience from field cases lead to the first signalament of the disease in Bosnia-Herzegovina, whereas during 2022 an outbreak of RMS was reported in a RAS system in Slovenia. For the first time RMS was described in juvenile rainbow trout as small as 35grs and MLO DNA was extracted from water samples. During the course of the project, we collected epidemiological data from more than 100 Italian rainbow trout farms through ad hoc questionnaire and map the presence of RMS across Italy. The present work could represent the foundation for future studies focused towards the complete molecular characterisation of MLO. Together with this, the use of ddPCR applied to MLO DNA and environmental DNA can certainly help to implement both biosecurity measures and lead to early diagnosis or even prediction of RMS outbreaks. Finally, studies of economic impact of this disease are needed to develop possible management strategies and options, especially from a commercial point of view.