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The present study aims to provide a specific protocol for transmission electron microscopy of a sample of skin of rainbow trout affected by red mark syndrome (RMS). The red mark syndrome is a skin disease that affects the rainbow trout (Oncorhynchus mykiss). The disease, probably due to the Midichloria-like organism infection, is not lethal, but morbidity can reach up to 60%, leading to significant economic impact associated with the downgrading of the commercial product, increased labor, and susceptibility to secondary infections. The ultrastructure analyses allowed an earlier study to identify the presence of scattered microorganisms characterized by an oval shape, mainly in the cytoplasm of the cells. The protocol developed in this study will be instrumental in visualizing the ultrastructure of the microorganism, which is probably responsible for red mark syndrome infection. [ABSTRACT FROM AUTHOR]

The present study aims to provide a specific protocol for transmission electron microscopy of a sample of skin of rainbow trout affected by red mark syndrome (RMS). The red mark syndrome is a skin disease that affects the rainbow trout (Oncorhynchus mykiss). The disease, probably due to the Midichloria-like organism infection, is not lethal, but morbidity can reach up to 60%, leading to significant economic impact associated with the downgrading of the commercial product, increased labor, and susceptibility to secondary infections. The ultrastructure analyses allowed an earlier study to identify the presence of scattered microorganisms characterized by an oval shape, mainly in the cytoplasm of the cells. The protocol developed in this study will be instrumental in visualizing the ultrastructure of the microorganism, which is probably responsible for red mark syndrome infection.

Keywords: digital droplet PCR; Midichloria-like organism; rainbow trout; recirculating aquaculture system; red mark syndrome EN digital droplet PCR Midichloria-like organism rainbow trout recirculating aquaculture system red mark syndrome 1889 1892 4 11/11/22 20221201 NES 221201 Red mark syndrome is a non-lethal skin disease, reported to affect farmed rainbow trout over 50 g worldwide (Metselaar et al., 2022). The manuscript by Oh et al. (2019) describes several cases of the disease in rainbow trout in South Korea, in which macroscopic lesions resembling RMS can be seen in juvenile rainbow trout weighed less than 30 g. [Extracted from the article]

Red mark syndrome (RMS) is a widespread skin disorder of rainbow trout in freshwater aquaculture, believed to be caused by a Midichloria-like organism (MLO). Here, we aimed to study the pathologic mechanisms at the origin of RMS by analyzing field samples from a recent outbreak through gene expression, MLO PCR, quantitative PCR, and a histopathological scoring system proposed for RMS lesions. Statistical analyses included a One-Way Analysis of Variance (ANOVA) with a Dunnett’s multiple comparisons test to assess differences among gene expression groups and a nonparametric Spearman correlation between various categories of skin lesions and PCR results. In short, the results confirmed the presence of a high quantity of 16S gene copy numbers of Midichloria-like organisms in diseased skin tissues. However, the number of Midichloria-like organisms detected was not correlated to the degree of severity of skin disease. Midichloria-like organism DNA was found in the spleen and head kidney. The spleen showed pathologic changes mainly of hyperplastic type, reflecting its direct involvement during infection. The most severe skin lesions were characterized by a high level of inflammatory cytokines sustaining and modulating the severe inflammatory process. IL-1 β, IL-6, IL-10, MHC-II, and TCR were upregulated in severe skin lesions, while IL-10 was highly expressed in moderate to severe ones. In the moderate form, the response was driven to produce immunoglobulins, which appeared crucial in controlling the skin disease’s severity. Altogether our results illustrated a complex immune interaction between the host and Midichloria-like organism.

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. • First description of the effect of temperature on the development of RMS pathology. • Macroscopic and microscopic classification of RMS lesions, including healing stages. • Good correlation between ddPCR values and macroscopic and microscopic lesion classification. • First time MLO visualization by using TEM in a experimental setting. [ABSTRACT FROM AUTHOR]