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1 Center for Fish Disease Research, Department of Microbiology, 220 Nash Hall, Oregon State University, Corvallis, OR 97331-3404, USA
2 Mycobacteriology Branch, Division of Tuberculosis Elimination, Centers for Disease Control and Prevention, Atlanta, GA, USA
3 Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, Scotland, UK
4 School of Veterinary Medicine, Ilam University, PO Box 69315-516, Ilam, Iran
Correspondence
Christopher M. Whipps
whippsc{at}onid.orst.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene, hsp65 and rpoB sequences of M. salmoniphilum strains are DQ866764–DQ866770, DQ866777–DQ866783 and DQ866790–DQ866797 (respectively DQ866768, DQ866777 and DQ866790 for the type strain).
A tree resulting from Bayesian analysis of rpoB sequences and a strict consensus tree from parsimony analysis of ITS sequences are available as supplementary material with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Decostere et al., 2004). However, several recent studies employing DNA sequence data as well as traditional methods of characterization have identified several additional Mycobacterium species that infect fishes (Heckert et al., 2001; Kent et al., 2004; Levi et al., 2003; Rhodes et al., 2003, 2005; Poort et al., 2006; Whipps et al., 2003). Delineation of piscine Mycobacterium species is important not only for animal health but also for human health, as some species are potentially zoonotic. As a case in point, M. marinum infections in humans are often associated with exposure to fish or aquaria (Aubry et al., 2002; Jernigan & Farr, 2000).
Mycobacteriosis in salmonid fishes has been reported in the literature, but the identity of the species responsible is elusive (Arakawa & Fryer, 1984; Ashburner, 1977; Brocklebank et al., 2003; Bruno et al., 1998; Ross, 1960, 1970). In 1960, Ross described the salmon mycobacterium as a unique species and proposed the name ‘Mycobacterium salmoniphilum’ (Ross, 1960). However, Gordon & Mihm (1959) identified these isolates as M. fortuitum. Penso et al. (1962) proposed that only one of the three strains was truly M. fortuitum, and Tsukamura et al. (1967) supported this finding with numerical classification analysis based upon 101 characters and suggested that they ‘not be named M. fortuitum’. Some of this taxonomic confusion may be explained by the observation that mycobacteria from salmonid fishes exhibit biochemical characteristics of both M. chelonae and M. fortuitum. The inability to distinguish these isolates confidently from M. fortuitum resulted in the omission of ‘M. salmoniphilum’ from the Approved Lists of Bacterial Names (Skerman et al., 1980).
Using a broad panel of biochemical analyses, Arakawa & Fryer (1984) tested additional mycobacterial isolates from salmon, found that they were most like M. chelonae and assigned them to ‘M. chelonae subsp. piscarium’. The subspecies name was later withdrawn (Arakawa et al., 1986), as serological analyses could not separate ‘M. chelonae subsp. piscarium’ from M. chelonae subsp. chelonae or M. chelonae subsp. abscessus. Nonetheless, these and other studies (Ashburner, 1977; Brocklebank et al., 2003; Bruno et al., 1998) attributed salmon mycobacteriosis to M. chelonae or an M. chelonae-like species.
The body of evidence suggests that these mycobacteria isolated from salmon represent a distinct species that is similar to M. chelonae or M. fortuitum. Therefore, we propose that the name ‘M. salmoniphilum’ suggested by Ross (1960) is valid and its revival warranted. We employed molecular and chemical methods in this study to resolve these conflicting data and to clarify the taxonomic position of mycobacteria isolated from salmon.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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or ‘M. chelonae subsp. piscarium’ (Arakawa & Fryer, 1984) and M. chelonae of Bruno et al. (1998) are listed in Table 1. Cultures were grown on solid-phase media normally used for mycobacteria, including Middlebrook 7H10, Löwenstein–Jensen, blood agar or MacConkey agar, and in Middlebrook 7H9 liquid broth, at 28–30 °C. Biochemical analyses were conducted using standard methods (Kent & Kubica, 1985).
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DNA isolation and PCR amplification.
DNA was extracted from cultures using the UltraClean microbial DNA isolation kit (MoBio Laboratories). Amplification of target genes by PCR was conducted using standard methods and primers of Kent et al. (2004), Poort et al. (2006), Selvaraju et al. (2005) and Adékambi et al. (2003). DNAs extracted from uninfected fish tissues were used as negative controls and were consistently negative. Amplification products were either purified directly with the QIAquick PCR Purification kit or excised from the gel and purified using the QIAgen Gel Extraction kit (Qiagen). Sequences were obtained directly from amplification products at the Nevada Genomics Center (Reno, NV, USA).
Phylogenetic analysis.
Nucleotide sequences of the small-subunit (SSU) rRNA gene from salmonid isolates were aligned to those of other rapidly growing type strains of mycobacteria. Mycobacterium tuberculosis H37RvT and Mycobacterium leprae TN were used as outgroups. With the sequences of the internal transcribed spacer (ITS) and heat-shock protein 65 (hsp65) and RNA polymerase 
-subunit (rpoB) genes, analyses focused only on the M. chelonae complex, i.e. M. chelonae, M. abscessus, Mycobacterium immunogenum, Mycobacterium massiliense and Mycobacterium bolletii, and other relevant sequences from BLAST matches on GenBank. M. fortuitum strains were used as the outgroup based on recent phylogenetic analyses (Adékambi & Drancourt, 2004; Devulder et al., 2005) and our preliminary analyses. Alignments were created with CLUSTAL_X (Thompson et al., 1997) and edited manually. Gaps were treated as a fifth character state. Parsimony analyses were conducted in PAUP*4.01 (Swofford, 1998). Maximum-parsimony analysis employed a heuristic search with 10 repetitions of random sequence addition and tree bisection and reconnection branch swapping. Bootstrap confidence values were calculated with a heuristic search using simple sequence addition and 100 replicates. Bayesian analyses were conducted in MrBayes (Ronquist & Huelsenbeck, 2003) under a general time-reversible (GTR) model, with 106 generations, tree sampling every 100 generations and a burn-in of 100 trees. Distance and maximum-likelihood analyses were also conducted in PAUP*4.01; they yielded equivalent results to parsimony and Bayesian analysis and are therefore not shown.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) as defined previously (Brown-Elliott & Wallace, 2002; Adékambi et al., 2004, 2006; Selvaraju et al., 2005). The result was consistent with previous studies that have identified these isolates as M. chelonae based primarily on biochemical analyses (Arakawa & Fryer, 1984; Brocklebank et al., 2003; Bruno et al., 1998). Some common characteristics within this group are a positive 3 day arylsulfatase test, negative nitrate reductase test and the absence of pigmentation. Differential biochemical characteristics include utilization of glucose or citrate and, highly significantly, a lack of growth at or above 37 °C (Table 2).
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), rpoB (Supplementary Fig. S1 available in IJSEM Online) and the ITS (Supplementary Fig. S2). These analyses consistently revealed that ‘M. salmoniphilum’ is monophyletic. Our analyses included a broad range of isolates, including those from the original description of ‘M. salmoniphilum’ (ATCC 13756, ATCC 13757 and ATCC 13758), those described as ‘M. chelonae subsp. piscarium’ by Arakawa & Fryer (1984), identical strains MT1890 and MT1900 (results for MT1890 only are shown) from Atlantic salmon incorrectly assigned to M. chelonae (Bruno et al., 1998) and sequences obtained independently of our study from isolate SOL 803 from salmon in a hatchery in eastern Canada (GenBank accession no. AY489137) (Fig. 1
). One of the original isolates of Ross (1960), ATCC 13756, was more like M. fortuitum by DNA sequencing, consistent with the observations of Penso et al. (1962) that one of Ross's isolates was M. fortuitum. These data also illustrated that more than one species of Mycobacterium may infect salmon. Indeed, Mycobacterium neoaurum has also been reported from salmon (Roberts, 2001).
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). Estimates of phylogeny of the M. chelonae complex varied depending on the gene analysed, as illustrated by the unstable position of ‘M. salmoniphilum’ in these trees. Analyses of the SSU rRNA gene and rpoB suggested that ‘M. salmoniphilum’ is sister to M. chelonae (Fig. 1 and Supplementary Fig. S1), whereas hsp65 analysis showed M. immunogenum to be the nearest neighbour (Fig. 2) and, with ITS analysis (Supplementary Fig. S2), M. abscessus was the sister species. Although a consensus cannot be established, all analyses supported ‘M. salmoniphilum’ as an independent lineage, as opposed to a subset of another species. Sequence similarity between members of the M. chelonae complex and ‘M. salmoniphilum’ was high in the SSU rRNA gene (98.6–99.6 %) and progressively lower in the hsp65 (92.0–96.7 %), rpoB (92.2–95.8 %) and ITS (86.3–95.9 %) sequences. Intraspecifically, DNA sequences of ‘M. salmoniphilum’ isolates from geographically distant locales in eastern and western North America, Australia and Europe possessed a minimum DNA sequence similarity for each gene region as follows: SSU rRNA gene (99.8 %), hsp65 (97.7 %), rpoB (98.0 %) and ITS (98.7 %).
Two-dimensional TLC of cell-wall mycolic acids of ‘M. salmoniphilum’ by Arakawa & Fryer (1984) suggested similarities to M. chelonae and M. abscessus, having 
and ' mycolates. Isolates lacked the epoxy mycolates found in M. fortuitum. HPLC patterns of mycolic acids confirmed that the study isolates shared some characteristics with the M. chelonae complex and with the M. fortuitum complex (Fig. 3). However, new HPLC analysis of cell-wall mycolic acids of ‘M. salmoniphilum’ isolates visually demonstrated two separate peak groups, lacking the middle peaks found in M. fortuitum (Fig. 3a; Butler & Kilburn, 1990). Although the HPLC patterns were similar to that of M. fortuitum (Fig. 3a), the number of mycolic acid peaks and the peak heights were different for ‘M. salmoniphilum’ (Fig. 3c). Thus, the distribution of mycolic acids supported the separation of the study isolates from species within the M. chelonae complex and M. fortuitum.
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was consistent with our findings, but the name ‘M. salmoniphilum’ has no current standing in nomenclature. Thus, in accordance with Rules 28a and 33a, b and c of the International Code of Nomenclature of Bacteria (Lapage et al., 1992), we propose the revival of this name based on our analysis of Ross's original isolates and other well-identified strains and ascribe them to Mycobacterium salmoniphilum sp. nov., nom. rev. We designate ATCC 13758T as the type strain based on the following criteria. Ross (1960) submitted three isolates to the ATCC; two were identified in this study as M. salmoniphilum (ATCC 13757 and ATCC 13758T) by DNA sequencing, and one was confirmed as M. fortuitum (ATCC 13756). Strain ATCC 13758T is also available from the DSMZ as DSM 43276T and therefore satisfies the ICSP requirement for deposit of type strains in two collections in different countries.
Description of Mycobacterium salmoniphilum (ex Ross 1960) sp. nov., nom. rev.
Mycobacterium salmoniphilum (sal.mo.ni.phi'lum. L. n. salmo -onis a salmon; Gr. adj. philos loving; N.L. neut. adj. salmoniphilum salmon-loving).
The description is as given by Ross (1960) and also follows the descriptions of ‘M. chelonae subsp. piscarium’ Arakawa & Fryer (1984) and ‘Salmoniphilum’ strains of M. fortuitum (Tsukamura et al., 1967). Cells are acid-fast bacilli, consistent with species of the genus Mycobacterium. Growth occurs on Middlebrook 7H10 agar, blood agar, MacConkey agar and Löwenstein–Jensen slants, forming cream-coloured, smooth, shiny colonies, visible after 4–6 days. Following incubation periods exceeding 10 days, colonies tend to appear waxy, with an irregular border and ‘fried egg’ morphology. Growth is observed at room temperature (20 °C) and with incubation at 28 to 30 °C. Weak or delayed growth may occur at 10 °C, and no growth is observed at or above 37 °C. Bacilli are generally slender and straight or slightly curved, with some short and thick. Dimensions range from 1 to 4 µm in length and 0.25 to 0.6 µm in width. Isolates have been recovered from infected salmonid fishes (although there may be other susceptible species), found throughout the viscera and predominantly in the kidney. Closely related species are members of the M. chelonae complex, i.e. M. chelonae, M. abscessus, M. immunogenum, M. massiliense and M. bolletii, sharing the characteristics of a positive 3 day arylsulfatase test, negative nitrate reductase test and the absence of pigmentation. Strains can be distinguished from these species by a positive test for glucose utilization and by DNA sequences. Sequence analysis of the SSU rRNA gene, hsp65, rpoB and ITS regions shows that isolates are phylogenetically and consistently distinct from other members of the M. chelonae complex. Mycolic acid HPLC patterns are visually distinct, forming two clusters of mycolates.
The type strain is strain SCT (=ATCC 13758T =DSM 43276T), isolated from Chinook salmon (Oncorhynchus tshawytscha) at the Spring Creek fish hatchery in Washington state, USA.
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ACKNOWLEDGEMENTS |
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