Article Figures & Data
Figures
- FIG. 1.
Phylogenetic relationships among Mycobacterium species derived from the 16S rRNA gene sequences, as constructed by maximum-parsimony analysis. The tree was rooted by using N. asteroides as the outgroup. Numbers on the branches indicate bootstrap proportions (100 replicates). Bar, 1.0 nucleotide replacement. Available GenBank accession numbers are shown.
- FIG. 2.
Phylogenetic relationships among Mycobacterium species derived from the hsp65 gene sequences (A) and the deduced sequences of the amino acids encoded by the Mycobacteriumhsp65 gene (B), as constructed by maximum-parsimony analysis. The tree was rooted by using N. asteroides as the outgroup. Numbers on the branches indicate bootstrap proportions (100 replicates). Bar, 1.0 nucleotide or amino acid replacement. Available GenBank accession numbers are shown.
- FIG. 3.
Enzyme restriction mapping. (A and B) A 614-bp PCR product from the 16S rRNA gene cleaved with restriction enzyme MslI. (A) Gel separation. (B) Restriction map. (C and D) A 796-bp product from the hsp65 gene cleaved with restriction enzyme DdeI. (C) Gel separation. (D) Restriction map. Lanes: M, 100-bp molecular size marker; 1, DL240490; 2, SR030597; 3, CC240299; 4, BB170200; 5, ATCC 927; 6, DLDK.
Tables
- TABLE 1.
Mycobacterium isolates and their hosts
Isolatea Host Originb Groupc Identity to GenBank accession no. for: 16S rRNA hsp65 16S rRNA hsp65 ATCC 927 Unspecified fish United States, marine, captive C CII AF456240 AF456470 DL240490 European sea bass (Dicentrarchus labrax) Israel, marine (RS), cultured A A AF456238 AF456468 DL150991 European sea bass (D. labrax) Israel, marine (MS), cultured A A AF456238 AF456468 DL045d European sea bass (D. labrax) Greece, marine (MS), cultured A A AF456241 AF456471 DL049d European sea bass (D. labrax) Greece, marine (MS), cultured F F AF456242 AF456472 CF030494 Butterfly fish (Chaetodon fasciatus) Israel, marine (RS), captive A A AF456238 AF456468 PP241194e Sea bream (Puntazzo puntazzo) Israel, marine (RS), cultured SO020195e Red drum (Sciaenops ocellatus) Israel, marine (RS), cultured SR250195 Rabbit fish (Siganus rivulatus) Israel, marine (RS), cultured A A AF456238 AF456468 DLDKf European sea bass (D. labrax) Denmark, marine, cultured C CII AF456240 AF456470 MC110595e Grey mullet (Mugil cephalus) Israel, marine (RS), cultured SA200695e Gilthead sea bream (Sparus aurata) Israel, marine (RS), cultured Hybrid 270995g Hybrid red sea bream (Pagrus major [female] × S. aurata [male]) Israel, marine (RS), cultured A AF456238 EA040995g White grouper (Epinephelus aeneus) Israel, marine (RS), cultured A AF456238 SR300397g Rabbit fish (S. rivulatus) Israel, marine (RS), wild A AF456238 SR030597 Rabbit fish (S. rivulatus) Israel, marine (RS), wild A A AF456238 AF456468 EI100398g Hawksbill sea turtle (Eretmochelys imbricata) Israel, marine (RS), captive A AF456238 CC240299 Koi (Cyprinus carpio) Israel, freshwater, cultured B B AF456239 AF456469 EA110499g White grouper (E. aeneus) Israel, marine (RS), cultured A AF456238 BB170200 Silver perch (Bidyanus bidyanus) Israel, freshwater, cultured B B AF456239 AF456469 SV300500 Lizard fish (Synodus variegatus) Israel, marine (RS), wild A A AF456238 AF456468 DL041200 European sea bass (D. labrax) Greece, marine (MS), cultured A A AF456238 AF456468 185-408h Golden steaty (Acipenser baeri) Holland, freshwater G G AF152558 j AF456473 185-409h Golden steaty (A. baeri) Holland, freshwater G G AF152558 j AF456473 S4i Snakehead (Channa striatus) Thailand, freshwater C CIII AF251565 j AF456474 S267i Snakehead (C. striatus) Thailand, freshwater C CIII AF251565 j AF456474 ↵ a Unless otherwise indicated, isolates were from Israel Oceanographic and Limnological Research Ltd., National Center for Mariculture, Eilat, Israel (the two first letters indicate the initials of the host genus and species; the digits indicate the date of isolation [daymonthyear]).
↵ b RS, Red Sea; MS, Mediterranean Sea.
↵ c According to the phylogenetic analysis as it appears in Fig. 1 and 2A.
↵ d Kindly provided by H. Nousias, Oceanos, Fish Health Center, Preveza, Greece.
↵ e Not included in the 16S rRNA and hsp65 gene sequence study.
↵ f Kindly provided by I. Dalsgaard, Fish Disease Laboratory, Danish Institute for Fisheries and Marine Research, Frederiksberg, Denmark.
g Not included in the hsp65 gene sequence study.
↵ h Kindly provided by O. L. M. Haenen and D. Bakker, Laboratory of Fish Diseases, Institute for Animal Science and Health, Lelystad, Holland.
↵ i Kindly provided by A. Adams, Institute of Aquaculture, University of Stirling, Stirling, Scotland.
↵ j Accession numbers are already available from GenBank.
- TABLE 2.
PCR primers
Gene Primer Positiona Sequenceb 16S rRNA 246c 1-20 F 5′-AGA GTT TGA TCC TGG CTC AG-3′ My1 61-78 F 5′-GGA AAG GTC TCT TCG GAG-3′ 414c 200-216 R 5′-CAT CCC ACA CCG CWA AAG-3′ 266c 593-612 R 5′-CAC GCY CAC AGT TAA GCY GT-3′ 614 593-614 F 5′-CTT AAC TGT GAG CGT GCG-3′ 1522c 1503-1522 R 5′-AAG GAG GTG ATC CAG CCG CA-3′ hsp65 HSF1 1-21 F 5′-GAT CCG GAG GAA TCA CTT CGC-3′ TB11d 167-186 F 5′-ACC AAC GAT GGT GTG TCC AT-3′ TB12d 588-607 R 5′-CTT GTC GAA CCG CAT ACC CT-3′ HS12F 588-607 F 5′-AGG GTA TGC GGT TCG ACA AG-3′ HS4F 1079-1099 F 5′-ACA GCG ACT CCG ACT ACG ACC-3′ M2e 1367-1383 R 5′-TTG AAG GCG ATC TGC TT-3′ HSR1 1628-1648 R 5′-TCA GAA ATC CAT GCC ACC CAT-3′ ↵ a According to GenBank accession no. AF456238 for the 16S rRNA gene and accession no. AF456468 for the hsp65 gene.
↵ b F, forward, R, reverse. Bold letters in primers sequences represent the following degeneracy: W, A or T; Y, C or T.
↵ c Designed by Böddinghaus et al. (2).
d Designed by Telenti et al. (31).
↵ e Designed by Plikaytis et al. (21).
- TABLE 3.
Mycobacterium sp. sequences available in the GenBank database compared to the sequences of our isolates
Gene study Isolate Origin Clustera GenBank accession no. 16S rRNA M. marinum Unknownb; identical to strain ATCC 927 C X52920 M. marinum Rainbow fish (Melanotaenia praecox); Taiwan C AF251565 Mycobacterium sp. strain M175 Striped bass (Morone saxatilis); United States C AY005147 M. ulcerans Human C X88926 M. bovis Strain BCG D M20940 M. tuberculosis Human; strain H37Rv D Z83862 M. avium Human E AF306455 M. avium subsp. paratuberculosis Strain ATCC 19698 E X52934 M. chlorophenolicum Freshwater lake sediment F X79094 Mycobacterium sp. strain Fuerth 1999 Human G AF152558 N. asteroidesc Strain ATCC 3306 H X57949 hsp65 M. ulcerans Human; Australia (isolate 93147172) CI AF456475 d M. avium subsp. paratuberculosis Ruminant E X74518 M. avium Unknownb E AF281650 M. tuberculosis Human; strain H37Rv D AL021932 M. bovis Strain BCG P3 D M17705 N. asteroides c Strain ATCC 3306 H AF352019 ↵ a According to the phylogenetic analysis as it appears in Fig. 1 and 2A.
↵ b Unspecified source.
↵ c Used as an outgroup.
↵ d Isolate was kindly provided by A. Sievers, Mycobacterium Reference Laboratory, Fairfield Hospital, Fairfield, Victoria, Australia. It was sequenced and submitted by us to GenBank.
