Effect of temperature on transfer of Midichloria-like organism and development of red mark syndrome in rainbow trout (Oncorhynchus mykiss)

Red mark syndrome (RMS) is a skin disease of rainbow trout (Oncorhynchus mykiss), believed to be bacterial and caused by a Midichloria-like organism (MLO). Based on reports from farm sites, clinical signs of RMS are not observed above 15–16 °C. Here we describe the onset and progression of RMS at different temperatures under controlled experimental settings. Nine 180 L tanks at 12 °C, 15.5 °C and 19 °C were used to cohabitate 162 specific pathogen free (SPF) rainbow trout with 24 RMS seeder fish. The fish were followed for 11 weeks post cohabitation (wpc) to monitor lesion development by visual assessment and by taking samples at 3, 8 and 11 wpc for histology from skin lesions and internal organs, samples of skin lesions and spleen for MLO detection by qPCR and by digital droplet PCR (ddPCR). Skin and spleen samples for MLO detection by transmission electron microscopy (TEM) were taken from selected cases. Macroscopic and microscopic skin lesions were classified based on 4 stages: mild, moderate, severe and healing. In short, the results showed that at 12 °C the development of skin lesions peaked in severity at 11 weeks post-cohabitation. At 15.5 °C the skin pathology was most severe at 8 weeks, and 3 weeks later skin lesions were in the healing phase or completely resolved. At 19 °C RMS skin lesions developed in just few cases and these lesions were predominantly classified as mild. Concentration of MLO DNA detected by both qPCR and ddPCR techniques seem to follow the same trend of the severity of skin lesions at 12 °C and 15.5 °C. MLO was visualized by using transmission electron microscopy on selected cases showing most severe skin lesions. This publication contains the first description of the effect of temperature on the development and resolution of RMS pathology and the presence of MLO; our study both describes for the first time the presence of MLO by using TEM on samples from a controlled experimental setting and it provides strong evidences that MLO is the etiological agent of RMS. Furthermore, a recently developed novel sensitive PCR technique (ddPCR) for detecting MLO was applied successfully. Additional insights about MLO involvement in RMS and details about RMS development illustrated here can represent the foundations for novel valuable strategies on RMS management and diagnostics.