T.E.M. AND BIOMOLECULAR DETECTION OF RICKETTSIALES IN TISSUES OF RAINBOW TROUT AND THEIR POTENTIAL ROLE AS RED MARK SYNDROME (RMS) ETIOLOGICAL AGENTS

RMS and US SD are skin disorders of farmed rainbow trout occurring in many European countries and US. The diseases aetiology is still uncertain. In spite of specific investigations identifying the presence of RLOs related DNA in tissues of US SD and RMS affected individuals (Lloyd et al., 2008; Metselaar et al., 2010), these pathogens have never been morphologically detected in samples. The present study represents the prosecution of previous researches carried out in the past years on Italian RMS outbreaks, finalized to comprehend the disease pathogenesis and causative agent. In detail we performed histological, T.E.M. and bimolecular analysis on numerous skin and spleen samples collected from RMS symptomatic fish, classified accordingly to the diagnostic criteria published by Oidtman et al. (2013). The histological evaluation underlined, in all skin lesions, the presence of the typical lymphocyte/macrophage infiltration involving various layers of this organ, still there was no evidence of microbial agents. T.E.M. observation revealed the presence of intra-cytoplasmic electron dense bacteria frequently surrounded by a clear halo, within macrophages, fibroblasts and erythrocytes. The microorganisms were oval or rod shaped, displaying a size ranging from 400 to 700 nm in length and 100 to 200 nm in width. They showed a thin cell wall lining an electron dense granular cytoplasm, that in some cases showed a thread like structure of DNA filled matrix located at the one pole of the bacterial body. Biomolecular insights allowed to detect a DNA related to Rickettsiales. The concomitant T.E.M. and PCR findings in RMS affected rainbow trout allow us to strongly suspect a bacteria belonging to Rickettsiales order as the causative agent of this disease. Further investigations are still necessary to clear the role of these bacteria in the pathogenesis of rainbow trout RMS.