Aetiology of red mark syndrome in rainbow trout (Oncorhynchus mykiss)

Abstract

Red mark syndrome (RMS) is a non-lethal skin condition, of unknown aetiology, affecting rainbow trout (Oncorhynchus mykiss) in the United Kingdom since 2003. It has now spread to 50% of the rainbow trout farms, resulting in great economic losses due to the downgrading of the product. There are also similar skin conditions in rainbow trout, for instance strawberry disease (SD) in the USA. As with RMS, the aetiological agent of this disease is also unknown. Several potential aetiological agents have been proposed, including a Rickettsia-like organism (RLO) in SD in the USA and Flavobacterium psychrophilum in RMS in the UK. The aim of the research presented here was to investigate the causative agent of RMS and to establish if there is a relationship between RMS and SD. An RLO was found to be associated with both RMS and SD-affected fish using immunohistochemistry (IHC) and polymerase chain reaction (PCR). The results of the IHC, together with the similarities in the pathology between the two conditions, suggest that RMS and SD are most likely the same disease (Chapter 2). In an attempt to isolate the RLO, F. psychrophilum or other suspected causative agents of RMS, several artificial bacteriological media, cell culture methods and novel techniques such as MagnaBind™ IgG beads (magnetic beads) were utilized. Although initial results appeared promising, no specific bacterial or viral agent was isolated using these methods. Transmission electron microscopy was used to analyse samples in an attempt to visualise any viruses and/or the RLO suspected of causing RMS (Chapter 3), but none were seen. Investigation into the involvement of both the RLO and F. psychrophilum in RMS using primary culture and IHC, together with the more advanced techniques of MALDI-TOF–MS and 16s rRNA gene sequencing, showed no association between F. psychrophilum and RMS. A quantitative PCR (qPCR), together with IHC, showed a positive correlation between the RLO and RMS-affected tissue, but this did not v distinguish between primary or secondary involvement of the organism. Results following analysis of samples using other assays, including ELISA and IHC, both using serum from naturally infected individuals, 16s rRNA gene PCR and bacterial isolation, were inconclusive, with methods requiring further optimisation for future use. The qPCR used in the study also needs to be fully optimised, as the results of a ring trial between three laboratories were considerably different (Chapter 4). Cohabitation challenges were conducted in the USA to investigate the involvement of the RLO in the early stages of SD. Clinical signs of SD were clearly evident in a small percentage of the cohabitated naïve fish. In most of these cases the DNA of the RLO could be detected, but again primary or secondary involvement could not be determined due to the small sample size (Chapter 5). In conclusion, the results from the analysis of samples by PCR, IHC with anti-F. psychrophilum PAbs, MALDI-TOF-MS and 16s rRNA gene sequencing indicate that F. psychrophilum is unlikely to be the causative aetiological agent of RMS. Although Koch’s postulates were not fulfilled, a strong correlation was obtained between the RLO and RMS-affected fish in the IHC, PCR and qPCR using RLO specific primers. It is unclear however, if the involvement of the RLO is as a primary or secondary pathogen. The RLO associated with RMS appears to have antigens in common with Piscirickettsia salmonis (from the results of the IHC), the causative agent of Salmon Rickettsial Syndrome, for which commercial vaccines are available, and should therefore be investigated as a form of mitigation for RMS, since the RLO has not yet been isolated and a traditional inactivated whole cell vaccine is not possible at this time. Efforts to isolate the RLO should continue and the involvement of other pathogens in RMS should be investigated further with new cutting edge techniques such as next generation sequencing or random multiplex (RT)-PCR to rule out viral involvement in the disease.