do salmon carry chlamydia

DNA sequencing and analysis.

PCR products and clones were selected based on the sample origin, i.e., Ireland or Norway, and the 16S rDNA target region, e.g., signature sequence, 806R, and near-full-length 16S. In each case, a minimum of four amplicons from several different samples and different PCR runs were designated for sequencing to account for intersample and intrasample variation. One clone from each separate cloning reaction was submitted for sequencing by primer walking in sense and antisense directions (HHMI Biopolymer/W. M. Keck Foundation Biotechnology Resource Laboratory). The nucleotide sequences of cloned PCR products were determined by assembling ABI sequence files for sense and antisense strands of each PCR product clone using Vector NTI version 7.0 (InforMax, Inc., Bethesda, Md.). Consensus sequences for 16S signature and 806R targets were constructed with respect to the sample source using multiple-sequence-alignment programs of DNAMAN (Lynnon Biosoft, Vaudreuil, Quebec, Canada) and Vector NTI version 7.0, in which base discrepancies were resolved by simple majority rulings. A 1,487-bp near-full-length 16S rDNA consensus sequence was constructed from amplicons from the year 2000 Norway samples and was aligned to all other consensus sequences to create an overall 16S rDNA consensus sequence of the CLB associated with epitheliocystis in proliferative gills, which was utilized in standard nucleotide-nucleotide BLAST searches of the National Center for Biotechnology Information databases (http://www.ncbi.nlm.nih.gov).

The consensus 16S rRNA gene sequence from the CLB was aligned with full-length 16S rRNA gene sequences selected from prior molecular systematic studies of members of the order Chlamydiales (9), Neochlamydia hartmannellae and endosymbionts of Acanthamoeba spp. (17), and members of the order Rickettsiales (6). Sequences downloaded for molecular phylogenetic analysis are listed with corresponding GenBank accession numbers in Table 2. Homology matrix analysis of the 16S rRNA gene sequence from the CLB with those of the organisms listed in Table 2 was performed using DNAMAN. Sequences for phylogenetic analyses were aligned, edited by visual inspection, and formatted using ClustalX version 1.81 (31; ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX/), and distance- and parsimony-based analyses were conducted using PAUP* version 4.0b10 for Macintosh (30). Parsimony-based analyses were executed using heuristic search settings that included random sequence addition, i.e., independent of the order of the input file, with 10 replicates per sequence addition and random trees used as starting points, tree-bisection-reconnection branch swapping, and collapsing of branches to create polytomies when maximum branch lengths were zero. Distance-based, i.e., minimum evolution, analyses were executed using heuristic search settings employing neighbor-joining to obtain starting trees and tree-bisection-reconnection branch swapping. In heuristic parsimony searches, the number of parsimony-informative characters was 671 of a total of 1,596. Estimates of confidence at nodes were obtained through 1,000 bootstrap replicates of heuristic searches (10).

Percent nucleotide identities between the 1,487-bp 16S rRNA gene sequence of “Candidatus Piscichlamydia salmonis” and those of the four families of Chlamydia, along with representatives of Rickettsia and Ehrlichia

No.	Species	% identity with species no.:
		37	36	35	34	33	32	31	30	29	28	27	26	25	24	23	22	21	20	19	18	17	16	15	14	13	12	11	10	9	8	7	6	5	4	3	2	1
1	Chlamydia muridarum MoPn [85718]	78	68	68	68	69	67	69	67	70	68	66	67	66	66	66	79	85	86	86	82	84	92	95	98	98	98	91	93	91	98	98	99	94	93	97	100	100
2	Chlamydia muridarum SFPD [U68437]	78	68	68	68	69	67	69	67	70	68	66	67	66	66	66	79	85	86	86	82	83	92	95	98	98	98	91	93	91	98	98	98	94	93	97	100
3	Chlamydia suis S45 [U73110]	78	68	68	68	69	67	68	66	69	68	66	67	65	67	66	80	84	86	86	82	83	91	94	97	97	97	90	92	90	97	97	97	93	94	100
4	Chlamydia suis R22 [U68420]	78	66	67	66	68	66	68	65	66	66	69	68	64	66	63	84	81	86	83	81	83	91	90	93	93	93	95	88	95	93	93	93	97	100
5	Chlamydia trachomatis L2/434/BU [U68443]	78	66	68	67	68	67	68	66	66	66	69	68	65	66	64	84	81	82	83	82	82	92	91	95	91	91	99	93	95	95	95	95	100
6	Chlamydia trachomatis A/Har-13 [D89067]	78	67	68	68	68	67	68	66	69	68	66	67	66	67	66	79	84	82	86	83	83	91	95	95	95	95	95	89	91	100	100	100
7	Chlamydia trachomatis B/TW-5/OT [D85719]	78	67	68	68	68	67	68	66	69	68	66	67	66	67	66	79	84	85	86	83	83	91	95	95	95	95	91	92	90	100	100
8	Chlamydia trachomatis DUW-3/CX [D85721]	78	67	68	68	68	67	68	66	69	68	66	67	66	67	66	79	84	85	86	83	83	91	95	95	95	95	91	92	91	100
9	Chlamydophila abortus EBA [U76710]	78	66	68	67	68	67	68	66	66	66	69	68	64	66	63	84	80	85	83	81	83	94	92	92	94	94	91	92	100
10	Chlamydophila psittaci MN [AB001784]	80	70	70	71	69	68	70	69	68	70	69	70	68	70	68	77	82	82	84	84	85	91	94	92	96	96	92	100
11	Chlamydophila psittaci NJI [U68419]	78	66	70	67	68	67	68	66	66	66	69	68	64	66	66	84	80	84	83	81	83	94	92	94	94	94	100
12	Chlamydophila caviae GPIC [D85708]	78	67	68	68	69	67	69	67	70	68	66	67	66	67	66	79	84	82	78	82	84	93	96	96	98	100
13	Chlamydophila felis FP Cello [D85706]	78	67	68	68	69	68	69	67	70	68	66	67	66	67	66	80	84	86	78	82	84	93	96	96	100
14	Chlamydophila peccrum E58 [D88317]	78	66	68	68	69	68	69	67	70	68	66	67	66	67	66	79	84	86	78	82	83	93	100	100
15	Chlamydophila peccrum IPA [D85716]	78	66	67	68	69	68	69	67	70	68	66	67	66	67	66	79	84	85	78	82	83	93	100
16	Chlamydophila pneumoniae N16 [U68426]	80	68	67	68	70	68	70	66	67	67	68	69	65	67	65	82	82	85	80	84	85	100
17	Endosymbiont UWC22 [AF083616]	81	70	69	70	70	69	70	69	66	70	70	70	69	70	66	82	84	88	81	92	100
18	Endosymbiont UWE1 [AF083614]	82	70	70	71	71	70	71	69	67	70	69	71	69	70	67	82	84	90	89	100
19	Neochlamydia hartmannellae [AF177275]	78	67	71	68	69	67	68	67	68	69	67	68	67	68	65	82	85	91	100
20	Parachlamydia acanthamoebae [Y07556]	79	67	68	68	69	68	69	67	70	68	66	67	66	67	66	84	87	100
21	Waddia chondrophila [AP042496]	77	66	66	67	68	67	68	65	69	66	64	66	65	66	64	80	100
22	Simkania negevensis [L27666]	77	65	66	65	66	65	67	64	65	65	68	67	64	65	62	100
23	Agent of withering syndrome in abalone [AF069062]	66	79	79	79	77	77	76	80	77	82	80	81	81	82	100
24	Ehrlichia chaffeensis [M73222]	69	83	83	83	81	83	82	85	80	92	91	92	98	100
25	Ehrlichia chaffeensis [U86664]	68	82	81	81	79	82	80	84	78	91	89	90	100
26	Ehrlichia sp. [AB074460]	71	83	84	83	81	82	82	83	79	91	92	100
27	Ehrlichia sp. ‘HGE agent’ [AF093788]	70	82	82	81	80	81	80	84	78	97	100
28	Ehrlichia sp. ‘HGE agent’ [U02521]	69	83	83	83	80	82	81	85	81	100
29	Ehrlichia risticii [M21290]	66	77	78	78	79	92	79	94	100
30	Ehrlichia sennetsu [M73225]	69	82	82	82	80	92	80	100
31	Orientia tsutsugamushi [D38622]	70	89	89	89	91	82	100
32	Necrickettsia helminthoeca [U12457]	70	80	81	81	82	100
33	Rickettsia typhi Wilmington [U12463]	70	96	96	96	100
34	Rickettsia rickettsii strain R [L36217]	70	98	98	100
35	Rickettsia sp. [AY158006]	70	98	100
36	Rickettsia sp. (Ixodes symbiont) [D84558]	69	100
37	“Candidatus Piscichlamydia salmonis”	100

Andrew Draghi IIDepartment of Pathobiology and Veterinary Science, University of Connecticut, Storrs, Connecticut,1 Electron Microscopy Laboratory, Department of Pathology, The University of Texas Medical Branch, Galveston, Texas,2 Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, Georgia,3 Department of Pathology, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire,4 Department of Pathology, New York University Medical Center, New York, New York5Find articles by

Received 2004 Mar 16; Revised 2004 May 27; Accepted 2004 Jul 5. Copyright © 2004, American Society for Microbiology

To characterize intracellular gram-negative bacteria associated with epitheliocystis in farmed Atlantic salmon (Salmo salar), gills with proliferative lesions were collected for histopathology, conventional transmission and immunoelectron microscopy, in situ hybridization, and DNA extraction during epitheliocystis outbreaks in Ireland and Norway in 1999 and 2000, respectively, and compared by ultrastructure and immunoreactivity to nonproliferative gills from Ireland archived in 1995. Genomic DNA from proliferative gills was used to amplify 16S ribosomal DNA (rDNA) for molecular phylogenetic analyses. Epitheliocystis inclusions from proliferative gills possessed variably elongate reticulate bodies, examples of binary fission, and vacuolated and nonvacuolated intermediate bodies, whereas inclusions in nonproliferative gills had typical chlamydial developmental stages plus distinctive head-and-tail cells. Immunogold processing using anti-chlamydial lipopolysaccharide antibody labeled reticulate bodies from proliferative and nonproliferative gills. 16S rDNA amplified directly from Irish (1999) and Norwegian (2000) gill samples demonstrated 99% nucleotide identity, and riboprobes transcribed from cloned near-full-length 16S rDNA amplicons from Norwegian gills hybridized with inclusions in proliferative lesions from Irish (1999) and Norwegian (2000) sections. A 1,487-bp consensus 16S rRNA gene sequence representing the chlamydia-like bacterium (CLB) from proliferative gills had the highest percent nucleotide identity with endosymbionts of Acanthamoeba spp. (order Chlamydiales). Molecular phylogenetic relationships inferred from 16S rRNA gene sequences using distance and parsimony indicated that the CLB from proliferative gills branched with members of the order Chlamydiales. “Candidatus Piscichlamydia salmonis” is proposed for the CLB associated with epitheliocystis from proliferative gills of Atlantic salmon, which exhibits developmental stages different from those identified in nonproliferative gills.

Epitheliocystis has been associated with heavy mortality and reduced growth of survivors in farmed Atlantic salmon (Salmo salar) (22). Ultrastructural studies of the epitheliocystis agent found in Atlantic salmon have revealed it to be an intracellular gram-negative coccoid bacterium with distinct developmental stages typical of bacteria of the order Chlamydiales (22). Epitheliocystis has been described in other salmonid hosts, e.g., juvenile steelhead trout (Oncorhynchus mykiss) (28) and cultured lake trout (Salvelinus namaycush) (3), as well as in a number of nonsalmonid species, including bluegill (Lepomis macrochirus) (16), striped bass (Morone saxatilis) (32), white perch (Morone americanus) (32), sea bream (Sparus aurata) (25), grey mullet (Liza ramada) (25), and cultured white sturgeon (Acipenser transmontanus) (14). Morphological studies of epitheliocystis agents in sea bream (S. aurata) have provided evidence for two distinct chlamydia-like developmental cycles associated with proliferative and nonproliferative host reactions (5). Although transmission electron microscopic examinations of intracellular inclusions have demonstrated that the agents of epitheliocystis in both salmonid and nonsalmonid hosts are gram-negative bacteria with developmental stages typical of members of the order Chlamydiales (5, 14, 22, 28, 32), the genetic relatedness of these bacteria has yet to be determined.

Sequence data from the rRNA operon have revised phylogenetic relationships between Chlamydia species and chlamydia-like bacteria (CLB) (7, 8, 9). Reclassifying chlamydial species on the basis of 16S rRNA gene sequence identity, 16S and 23S ribosomal DNA (rDNA) sequences, and phenotypic characterization is considered by some to be the best means of taxonomically categorizing chlamydiae (24). Based on this approach, species within the family Chlamydiaceae have 16S rRNA gene sequences that are >90% identical (26), whereas chlamydia-like bacteria, defined as obligate intracellular bacteria having reticulate (RBs) and elementary bodies (EBs) characteristic of chlamydia, have been shown to have >80% 16S rRNA gene sequence identity, e.g., Simkania negevensis strain Z (19) and “Candidatus Parachlamydia acanthamoebae” (1).

Unlike morphologically similar chlamydia-like bacteria, such as S. negevensis strain Z (19) and endosymbionts of Acanthamoeba spp. (11), the agents of epitheliocystis from fish have never been successfully cultured in vitro to facilitate genetic studies. Neither antigenic reactivity nor 16S rDNA sequence data have been obtained to further a molecular characterization of a chlamydia-like bacterium from a salmonid host. The objectives of this study were to compare the ultrastructures and immunoreactivities of developmental stages of inclusions from proliferative and nonproliferative gill lesions of farmed Atlantic salmon and to perform molecular phylogenetic analyses of 16S rDNA sequence data generated directly from proliferative gill lesions.

Samples of gill from farmed Atlantic salmon (S. salar) were collected at separate times by the staff of a multinational aquaculture company as part of its health surveillance program during periods of increased mortality, which were confirmed as outbreaks of epitheliocystis by histopathologic analysis of gill sections. Two sets of gill arches from 20 Atlantic salmon were submitted as pooled samples from one site in Ireland in 1999, and two sets of gill arches from 25 Atlantic salmon were submitted as individually identified samples from one site in Norway in 2000. One set of tissue samples from each location was submitted fixed by immersion in 10% formalin for histopathologic, electron microscopic, and in situ hybridization studies. A second set of tissues was submitted in 70% ethanol in the case of the Irish samples or immersed directly in tissue lysis buffer (ATL Buffer; QIAGEN Inc., Chatsworth, Calif.) in the case of the Norwegian samples for DNA extraction and PCR studies. In addition, paraffin- and resin-embedded gill samples from Atlantic salmon from Ireland accessioned in 1995 and processed for histopathology and transmission electron microscopy, respectively, were retrieved from departmental archives.

Formalin-fixed gill samples were trimmed to fit plastic cassettes, processed routinely for paraffin embedding, sectioned at 4 μm, mounted on glass slides, and stained with hematoxylin and eosin (HE) according to standard histologic techniques (29). Tissue sections were examined by light microscopy to identify histopathologic lesions and inclusions of epitheliocystis based on previous descriptions (22).

Do I Have Chlamydia? Symptoms of Chlamydia

FAQ

FAQ

Can fish carry chlamydia?

Aided by advances in molecular detection and typing, recent years have seen an explosion in the description of these epitheliocystis-related chlamydial pathogens of fish, significantly broadening our knowledge of the genetic diversity of the order Chlamydiales.

Do salmon carry diseases?

Bacteria have been found to be the most important agents of disease in the several species of Pacific salmon. Kidney disease, due to a small, unnamed Gram-positive diplobacillus, causes serious mortalities in young salmon reared in hatcheries. The disease has also been found in wild fish.

What animals carry chlamydia?

What parasites do salmon carry?