Detection and characterisation of aquatic Mycobacterium spp.
Mvcohacterium spp, the etiological agent of mycobacteriosis, has recently been responsible for serious infections in two economically important fish species cultured in Thailand; snakehead (Channa striata) and Siamese fighting fish (Berta splendens). Attempts to detect and identify the pathogens to species level, in fish tissue and the environment, have so far been unsuccessful, mainly due to the poor levels of sensitivity and specificity of the detection methods used, based on conventional bacteriology and histology. In this study, a variety of novel techniques were developed and used for more effective identification of Mycobacterium spp., including monoclonal antibody-based assays, DNA-based techniques and mass spectrometry. A monoclonal antibody (Mab 8F7) probe was developed against M. marinum, which was successfully used to identify M. marinum in infected fish tissue by immunohistochemistry (IHC), and from pure bacterial cultures by enzyme-liked immunosorbent assay (ELISA). The molecular-based techniques employed to detect the pathogen included in situ hybridisation (ISH), polymerase chain reaction (PCR) and reverse cross blot hybridisation. The PCR was developed using primers available from the literature which amplified mycobacterial 16S rDNA. The products of the reaction were identified to species level by PCR-reverse cross blot hybridisation. M. inarinum, M. fortuitum and M. chelonae were identified using this method. The same primers as those used in the PCR, were used as probes in ISH to identify Mycobacterium spp to genus level in infected fish tissues. spectrometry. A range of Mvcohocterium spp. isolated from fish located in different geographical regions were identified and characterised using Mab 8F7, pyrolysis mass spectrometry (PyMS) and PCR-reverse cross blot hybridisation and PyMS analysis showed that three distinct groups of mycobacteria were involved in mycobacteriosis in Thailand and Israel. The groups were clustered around either type strains M. fortuitum-M. chelonae or M. marinum, or around an unspeciated Mycobacterium spp. The unspeciated isolates were identified as M. marinum by PCR analysis and were mainly isolated from fish cultured in Israel. M. marinum from Israel and Thailand appeared to be different from each other since the isolates from Thailand reacted positively with Mab 8F7, whereas isolates obtained from fish in Israel were negative. PCR-reverse cross blot hybridisation was used to establish the identity of Mycobacterium spp involved in mycobacteriosis outbreaks affecting Siamese fighting fish and snakehead fish in Thailand. PCR was also utilised to analyse environmental samples taken from these farm sites. Siamese fighting fish farmers are known to suffer from skin lesions caused by Mycobacterium spp and therefore biopsies were taken from the farmers for analysis by PCR-reverse cross blot hybridisation. Analysis revealed that two species, M. fortuitum and M. marinum, were involved in the mycobacterial infections observed in both fish species. M. fortuitum and M. marinum were also both found in environmental samples including water, sediment and fish food. However, M. fortuitum was the isolate most frequently found. Skin lesions were only observed amongst the Siamese fighting fish farmers, while the snakehead fish farmers did not seem to be effected. Analysis of the biopsies from the skin lesions by PCR reverse cross blot hybridisation revealed that M. fortuitum was the main etiological agent associated with these lesions.