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

Abstract

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°C for 2 months, Mycobacterium sp. was isolated from 25% of the fish. The isolates were identified by standard biochemical tests. A 441 bp 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.U.S.

Detection of Mycobacterium marinum, M. peregrinum, M. fortuitum and M. abscessus in aquarium fish

“…flavescens , M. gordonae , M. haemophilum , M. kansasii , M. monteforense , M. neoaurum , M. peregrinum , M. salmoniphilum , M. scrofulaceum , M. simiae , M. shottsii and M. terrae (Beran, Matlova, Dvorska, Svastova, & Pavlik, ; Chang et al., ; Guz et al., ; Levi et al., ; Marumo et al., ; Pate, Jencic, Zolnir‐Dovc, & Ocepek, ; Řehulka, Kaustová, & Řehulková, 2006; Rhodes et al., ; Whipps, Dougan, & Kent, ; Zanoni, Florio, Fioravanti, Rossi, & Prearo, 2008; Zerihun, Berg, Lyche, Colquhoun, & Poppe, ). The zoonotic nature of mycobacteriosis and massive economic losses in the aquaculture industry highlight the need for rapid identification and treatment of mycobacterial infections (Shukla, Shukla, Sharma, & Kumar, ; Szmygin‐Milanowska et al., ). Matrix‐assisted laser desorption ionization‐time of flight mass spectrometry (MALDI‐TOF‐MS) has been used for accurate and rapid identification of mycobacteria (Hettick et al., ; Saleeb, Drake, Murray, & Zelazny, ; Shitikov et al., ).…”

“…The most important species causing mycobacteriosis in fish are non-tuberculous mycobacteria, that is M. chelonae, M. fortuitum and M. marinum (Ross, & Brancato, 1959;Decostere, Hermans, & Haesebrouck, 2004;Han et al, 2013;Kent et al, 2004;Lescenko et al, 2003;Najiah, Lee, Noorasikin, Nadirah, & Lee, 2011;Novotny et al, 2010;Sevim, Ozer, & Rad, 2015;Slany, Makovcova, Jezek, Bodnarova, & Pavlik, 2014) (Beran, Matlova, Dvorska, Svastova, & Pavlik, 2006;Chang et al, 2006;Guz et al, 2013;Levi et al, 2003;Marumo et al, 2010;Pate, Jencic, Zolnir-Dovc, & Ocepek, 2005;Rehulka, Kaustov a, & Rehulkov a, 2006;Rhodes et al, 2003;Whipps, Dougan, & Kent, 2007;Zanoni, Florio, Fioravanti, Rossi, & Prearo, 2008;Zerihun, Berg, Lyche, Colquhoun, & Poppe, 2011). The zoonotic nature of mycobacteriosis and massive economic losses in the aquaculture industry highlight the need for rapid identification and treatment of mycobacterial infections (Shukla, Shukla, Sharma, & Kumar, 2014;Szmygin-Milanowska et al, 2016).…”

Investigation and management of an outbreak of multispecies mycobacteriosis in Australian lungfish (Neoceratodus fosteri) including the use of triple antibiotic treatment

“…; Shukla et al . ) . Mycobacterium marinum , M. chelonae and M. fortuitum are among eight species of mycobacteria to have been identified from PME and antemortem lesions by culture and PCR identification in individual infections in many fish species at ZSL London Zoo's aquarium over the last 18 years (Routh ; Macgregor pers.…”

“…have been isolated in both freshwater and saltwater water inlets in the aquarium (Routh 2013) and may have been a source of infection for the lungfish. Apparently healthy fish are known to carry various NTM (Beran et al 2006;Zanoni et al 2008;Kent et al 2009;Rose et al 2013;Shukla et al 2014). Mycobacterium marinum, M. chelonae and M. fortuitum are among eight species of mycobacteria to have been identified from PME and antemortem lesions by culture and PCR identification in individual infections in many fish species at ZSL London Zoo's aquarium over the last 18 years (Routh 2013;Macgregor pers.…”

Occurrence of <i>Mycobacterium</i> spp. in ornamental fish

“…However, due to the difference in clinical significance of NTM, species identification of mycobacterial isolates is necessary. Traditional methods, such as biochemical tests, fail to provide a precise identification of closely related NTM species [18]. Furthermore, molecular methods, such as hybridization DNA probe assays, 16S rRNA gene multiplex PCR, or PCR restriction fragment length polymorphism analysis (PRA) for identification of nontuberculous mycobacteria, might fail to distinguish closely related species [19].…”