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Immunohistochemical and Taqman real‐time PCR detection of mycobacterial infections in fish
Author(s) -
Zerihun M A,
Hjortaas M J,
Falk K,
Colquhoun D J
Publication year - 2011
Publication title -
journal of fish diseases
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.819
H-Index - 85
eISSN - 1365-2761
pISSN - 0140-7775
DOI - 10.1111/j.1365-2761.2010.01231.x
Subject(s) - biology , mycobacterium chelonae , microbiology and biotechnology , mycobacterium , rpob , mycobacterium marinum , mycobacterium fortuitum , nontuberculous mycobacteria , polymerase chain reaction , taqman , real time polymerase chain reaction , bacteria , 16s ribosomal rna , gene , biochemistry , genetics
Real‐time PCR and immunohistochemistry (IHC) assays were developed to detect fish mycobacterial infections at the genus level, based on the RNA polymerase β subunit ( rpoB ) gene and polyclonal anti‐ Mycobacterium rabbit serum, respectively. The PCR assay positively identified a number of pathogenic mycobacteria including Mycobacterium abscessus , M. avium ssp. avium , M. bohemicum , M. chelonae ssp. chelonae , M. farcinogenes , M. flavescens , M. fortuitum ssp. fortuitum , M. gastri , M. gordonae, M. immunogenicum , M. malmoense , M. marinum , M. montefiorense , M. phlei, M. phocaicum , M. pseudoshottsii , M. salmoniphilum , M. senegalense , M. shottsii , M. smegmatis , M. szulgi and M. wolinskyi . A detection limit equivalent to 10 2 cfu g −1 was registered for M. salmoniphilum‐ infected fish tissue. The IHC precisely localized both free and intracellular mycobacteria in tissues and detected mycobacterial infections down to 10 2 cfu g −1 tissue. Both assays were found to be more sensitive than Ziehl–Neelsen (ZN) staining, where the detection limit was below 8 × 10 3 cfu g −1 tissue. Although specificity testing of the real‐time PCR against a panel of non‐ Mycobacterium spp . revealed a degree of cross‐reaction against pure DNA extracted from Nocardia seriolae and Rhodococcus erythropolis, no cross‐reactions were identified (by either real‐time PCR or IHC) on testing of formalin‐fixed paraffin‐embedded (FFPE) tissues confirmed to be infected with these bacteria. The broad applicability of both assays was confirmed by analysis of FFPE tissues from a range of fish species infected with diverse Mycobacterium spp. The results indicate that both assays, alone or in combination, constitute sensitive tools for initial, rapid diagnosis of mycobacteriosis in fish. This should in turn allow rapid application of more specific studies, i.e. culture based, to identify the specific Mycobacterium sp. involved.