Identification of Mycobacterium species from apparently healthy freshwater aquarium fish using partial sequencing and PCR- RFLP analysis of heat shock protein ( hsp65) gene

The present study analysed the incidence of mycobacteria in apparently healthy looking freshwater aquarium fish in Uttar Pradesh (State), India. Sixty fish belonging to eight different species were collected from six aquarium shops in different cities and processed for isolation of Mycobacterium species. Using the initial protocol of decontamination of tissue homogenates (with 1N HCl & 2N NaOH) and incubation at 30 degrees C for 2months, Mycobacterium sp. was isolated from 25% of the fish. The isolates were identified by standard biochemical tests. A 441bp fragment of the hsp65 gene was amplified and digested by two fastdigest restriction enzymes, BstEII and HaeIII. Digested products were analysed using agarose gel electrophoresis. Sequencing of amplified fragments of the hsp65 gene was also performed. Isolates were identified as: five isolates of M.abscessus, three M.gordonae, two M.fortuitum, two M.conceptionense, two M.parascrofulaceum, and one isolate of M.senegalense. Mycobacterial incidence in apparently healthy looking freshwater aquarium fish is dreadful and the study is relevant because of the mycobacterial diversity related to aquarium fish and its zoonotic importance. All Mycobacterium species isolated in this study are well known pathogens in humans as well as fish.