can salmon get chlamydia

You can find their work in the Electron Microscopy Laboratory, Department of Pathology, The University of Texas Medical Branch, Galveston, Texas, the Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, Georgia, the Department of Pathology at Dartmouth-Hitchcock Medical Center in Lebanon, New Hampshire, and the Department of Pathology at New York University Medical Center in New York, New York.

You can find their work in the Electron Microscopy Laboratory, Department of Pathology, The University of Texas Medical Branch, Galveston, Texas, the Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, Georgia, the Department of Pathology at Dartmouth-Hitchcock Medical Center in Lebanon, New Hampshire, and the Department of Pathology at New York University Medical Center in New York, New York.

You can find their work in the Electron Microscopy Laboratory, Department of Pathology, The University of Texas Medical Branch, Galveston, Texas, the Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, Georgia, the Department of Pathology at Dartmouth-Hitchcock Medical Center in Lebanon, New Hampshire, and the Department of Pathology at New York University Medical Center in New York, New York.

You can find their work in the Electron Microscopy Laboratory, Department of Pathology, The University of Texas Medical Branch, Galveston, Texas, the Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, Georgia, the Department of Pathology at Dartmouth-Hitchcock Medical Center in Lebanon, New Hampshire, and the Department of Pathology at New York University Medical Center in New York, New York.

You can find their work in the Electron Microscopy Laboratory, Department of Pathology, The University of Texas Medical Branch, Galveston, Texas, the Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, Georgia, the Department of Pathology at Dartmouth-Hitchcock Medical Center in Lebanon, New Hampshire, and the Department of Pathology at New York University Medical Center in New York, New York.

You can find their work in the Electron Microscopy Laboratory, Department of Pathology, The University of Texas Medical Branch, Galveston, Texas, the Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, Georgia, the Department of Pathology at Dartmouth-Hitchcock Medical Center in Lebanon, New Hampshire, and the Department of Pathology at New York University Medical Center in New York, New York.

You can find their work in the Electron Microscopy Laboratory, Department of Pathology, The University of Texas Medical Branch, Galveston, Texas, the Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, Georgia, the Department of Pathology at Dartmouth-Hitchcock Medical Center in Lebanon, New Hampshire, and the Department of Pathology at New York University Medical Center in New York, New York.

You can find their work in the Electron Microscopy Laboratory, Department of Pathology, The University of Texas Medical Branch, Galveston, Texas, the Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, Georgia, the Department of Pathology at Dartmouth-Hitchcock Medical Center in Lebanon, New Hampshire, and the Department of Pathology at New York University Medical Center in New York, New York.

To learn more about the gram-negative bacteria that live inside cells of farmed Atlantic salmon (Salmo salar), gills with growing sores were taken 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. Then, they were compared by ultrastructure and immunoreactivity to gills from Ireland that did not have any growths from 1995. Genomic DNA from proliferative gills was used to amplify 16S ribosomal DNA (rDNA) for molecular phylogenetic analyses. Epitheliocystis inclusions from proliferative gills had varyingly long reticulate bodies, examples of binary fission, and vacuolated and nonvacuolated intermediate bodies. Inclusions from nonproliferative gills, on the other hand, had normal chlamydial developmental stages plus unique head-and-tail cells. Immunogold processing using anti-chlamydial lipopolysaccharide antibody labeled reticulate bodies from proliferative and nonproliferative gills. Amplification of %2016S%20rDNA directly from Irish (1999) and Norwegian (2000) gill samples confirmed 99% nucleotide identity, and riboprobes were synthesized from cloned near-full-length %2016S%20rDNAamplicons from Norwegian gills hybridized with inclusions in proliferation-prone lesions from Irish (1999) and Norwegian (2000) sections. A consensus 16S rRNA gene sequence of 1,487 bases that encodes the chlamydia-like bacterium (CLB) from proliferating gills shared the most nucleotides with endosymbionts of Acanthamoeba spp. (order Chlamydiales). By using distance and parsimony to figure out molecular phylogenetic relationships between 16S rRNA gene sequences, we saw that the CLB from proliferating gills branched with Chlamydiales members. “Candidatus Piscichlamydia salmonis” is a name for the CLB found in epitheliocystis from Atlantic salmon gills that are proliferating. This type of gill shows different developmental stages than gills that are not proliferating.

Epitheliocystis has been linked to a lot of deaths and slower growth in farmed Atlantic salmon (Salmo salar) (22). Ultrastructural studies of the epitheliocystis agent found in Atlantic salmon have shown that it is a gram-negative coccoid bacterium that lives inside cells and goes through the typical stages of development for bacteria in 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). It has also been found in 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). Transmission electron microscopy studies of intracellular inclusions have shown that the epitheliocystis-causing agents in both salmonid and nonsalmonid hosts are gram-negative bacteria that develop in a way typical of Chlamydiales (5, 14, 22, 28, 32). However, it is still not known how genetically related these bacteria are to each other.

The phylogenetic relationships between Chlamydia species and chlamydia-like bacteria (CLB) have been changed by sequence data from the rRNA operon (7, 8, 9). Some people think that the best way to taxonomically group chlamydiae is to reclassify them based on 16S rRNA gene sequence identity, 16S and 23S ribosomal DNA (rDNA) sequences, and phenotypic characterization. This method shows that species in the Chlamydiaceae family have 16S rRNA gene sequences that are 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. It is not possible to get antigen reactivity or 16S rDNA sequence data to help with the molecular characterization of a chlamydia-like bacterium from a salmonid host. We wanted to find out how the ultrastructures and immune responses of developmental stages of inclusions from proliferative and nonproliferative gill lesions of farmed Atlantic salmon were different. We also wanted to do 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 different times by the staff of a multinational aquaculture company as part of its health surveillance program. This was done during times when there was a lot of death, which was confirmed to be epitheliocystis outbreaks by histopathologic analysis of gill sections. In 1999, two sets of gill arches from 20 Atlantic salmon were sent as combined samples from the same site in Ireland. In 2000, two sets of gill arches from 25 Atlantic salmon were sent as separate samples from the same site in Norway. One set of tissue samples from each site was fixed by immersion in formalin for histopathologic, electron microscopy, and in situ hybridization studies. A second set of tissues was put in 70% ethanol for the Irish samples or directly in tissue lysis buffer (ATL Buffer; QIAGEN Inc.). , Chatsworth, Calif. ) in the case of the Norwegian samples for DNA extraction and PCR studies. Also, paraffin- and resin-embedded gill samples from Atlantic salmon from Ireland that were collected in 1995 and processed for histopathology and transmission electron microscopy were obtained from the department’s archives.

Using formalin to fix gill samples, they were cut to fit plastic cassettes, put through the normal steps for paraffin embedding, sectioned at 4 μm, mounted on glass slides, and stained with hematoxylin and eosin (HE) as per standard histologic procedures (29). Light microscopy was used to look at tissue sections and find histopathologic lesions and epitheliocystis inclusions based on what had been described before (22).

In situ hybridization using riboprobes.

In situ hybridization was accomplished using riboprobes generated by in vitro transcription of the cloned ∼1. They used a dual promoter vector (pCRII TOPO Vector; Invitrogen Corp) and followed the steps outlined by Brown (4) to make a 5-kb 16S RNA gene sequence from gills that were infected with the CLB. ) and Sp6/T7 DIG RNA labeling kit (Roche Applied Science, Indianapolis, Ind. ). Plasmid inserts were confirmed and orientations were determined by DNA sequencing. The full-length 16S rRNA gene of Mycobacterium marinum was copied in the same way in a lab to serve as a probe sequence control. Transcribed riboprobe concentrations were assessed by dot blot analysis.

Riboprobes were used in nonisotopic in situ hybridization experiments based on procedures described by Gan et al. (13) and Brown (4). Atlantic salmon gill tissue that wasn’t infected and came from somewhere other than Ireland or Norway was used as the negative tissue control. We looked at HE-stained gill sections under a light microscope to see how many and where the inclusions were located. Then, 4 μm tissue sections were mounted on ProbeOn Plus glass slides (Fisher Scientific, Fairlawn, N.J.) without any staining. J. ). Unstained sections were heated to 70°C for 10 min, deparaffinized in three 3-min washes with xylene (Sigma, St. Louis, Mo. ), and rehydrated for 15 min at RT. Pieces were broken down with 20 ng of proteinase K/μl at 37°C for 15 minutes. They were then covered with prehybridization solution (5— SSC [1— SSC is 0]). 15 M NaCl plus 0. 015 M sodium citrate], 5% blocking reagent, 48% formamide, 0. 02% sodium dodecyl sulfate [SDS], 0. 1% N-lauroylsarcosine) for 1 h at 37°C. To denature the target DNA, the slides were heated to 95°C for 10 minutes. Then, about 100 μl of hybridization solution (35 ng of digoxigenin-labeled riboprobe per 100 μl of prehybridization solution) was put on each slide, along with a glass coverslip. The slides were left to hybridize overnight at 42°C in a humidified slide moat (Fisher Scientific). Following hybridization, the slides were washed in 2%C3%97%20SSC%20with%201%%20SDS%20at%2050%C2%B0C%20for%2020%20min, then in 1%C3%97%20SSC%20with%200 1% SDS at 50°C for 20 min and then 1× SSC for 10 min at RT and 0. 1× SSC for 15 min at RT. Hybridization signals were developed by washing sections in buffer I (0. 1 M Tris, 0. 15 M NaCl, pH 7. 5) for 5 minutes and then putting the sections in an incubator for 2 hours at 37°C in a humid room with buffer I containing 2% sheep serum and anti-digoxigenin Fab fragments conjugated to alkaline phosphatase at a dilution of 1:275 (Roche Applied Science). The slides received one 15-min wash in buffer I and then were washed twice in TBST (0. 15 M NaCl, 2. 7 mM KCl, 25 mM Tris, 0. 05% Tween 20, pH 7. 6) for 5 min. The signal was developed using the DAKO (Carpinteria, Calif. ) Fuchsin-Substrate System with levamasole; the slides were counterstained and coverslipped according to the instructions of the manufacturer.

DNA extraction, amplification, and cloning.

Genomic DNA from ethanol-fixed or fresh gill tissue was extracted using the DNeasy extraction system (QIAGEN Inc. ) following the manufacturer’s protocol for mouse tails, with the following change: gill tissues in tissue lysis buffer were heated to 97 to 100°C for 15 minutes to help release bacterial DNA. Then, the lysis solutions were cooled to 55°C, proteinase K was added, and the lysis was left to happen overnight. Eluates were assessed by either spectrophotometric or fluorometric methods (VersaFluor Fluorometer; Bio-Rad, Inc. , Hercules, Calif. ). The DNA was aliquoted and stored at 4°C for immediate use or frozen at −80°C for future experiments. Negative control genomic DNA from Atlantic salmon was taken from skeletal muscle that was bought in stores or from whole-blood samples from Atlantic salmon that were not sick (thanks to John Coll, Fish Health Center, Northeast Fishery Center, U.S. S. Fish and Wildlife Service, Lamar, Pa. ). Muscle and blood were chosen as sources of genomic DNA from Atlantic salmon because they are not likely to contain chlamydia-like or other bacteria that could contaminate the DNA. DNA from a culture of Chlamydia trachomatis (ATCC VR1477; American Type Culture Collection, Manassas, VA) was used as a positive control. ). We checked the nucleic acid samples for amplifiable DNA by PCR using primers 18e and 18i or 18e and 18g on host 18S rDNA (15).

Oligonucleotide primers used to amplify 16S rDNA of C. trachomatis and the CLB linked to epitheliocystis from growing gills were picked to amplify three overlapping regions of the 16S rRNA gene that got longer as you went up. These included regions for finding the 16S rRNA gene signature sequence of Chlamydiales (9) or mixes of Chlamydiales 16S rRNA gene primers (9) and eubacterial primers (12, 27) (Table). The primers were synthesized by Invitrogen Corp. (Carlsbad, Calif. ).

Each 50-μl reaction mixture had 100 to 200 ng of sample DNA, 5 μl of 10× QIAGEN buffer (100 mM Tris-HCl, pH 8), and this was used to copy all 16S rDNA. 3, 500 mM KCl, 15 mM MgCl2, 0. The sample included 1% gelatin, 20% CE%BCM deoxynucleoside triphosphates, 2020 pmol of forward and reverse primers/%CE%BCl, and 1% U of QIAGEN HotStar Taq polymerase using a Perkin-Elmer model 2400 thermal cycler from Applied Biosystems in Foster City, California. ). Three different cycling protocols were employed to amplify the three overlapping 16S rDNA targets. For the 16S signature sequence, a touchdown PCR protocol (23) was used to limit secondary priming. The reactions began with 15 minutes of incubation at 94°C. There were then 40 cycles of denaturation at 94°C for 45 seconds, primer annealing for 45 seconds, and extension at 72°C for 45 seconds. Annealing temperatures started at 66°C and went down by 1°C every third cycle until they reached 61°C, which is where the last 25 cycles were done. After 40 cycles, a 7-min extension step at 72°C was performed. For the 806R protocol, PCR started with 15 minutes at 94°C. There were then 40 cycles, with denaturation at 94°C for 30 seconds, annealing at 55°C for 45 seconds, and extension at 72°C for 45 seconds. The last extension took 7 minutes. For almost full-length 16S rRNA amplicons, PCR started with 15 minutes at 94°C. There were then 40 cycles, with denaturation at 94°C for 40 seconds, annealing at 58°C for 40 seconds, and extension at 72°C for 45 seconds. There was then a final 7-minute extension step. The PCR products were separated using polyacrylamide or 2% agarose gel electrophoresis. They were then visualized using ethidium bromide staining and UV transillumination, and they were digitally recorded using a Stratagene (La Jolla, California) ) Eagle Eye II-Still Video System. Reactions that yielded amplicons were repeated; samples that amplified twice were considered for DNA sequence analysis.

The PCR products were taken out of agarose gels and cleaned up using a QIAGEN Mini-Elute Gel Extraction kit or a PCR Purification kit. The 16S signature sequence PCR products that had been cleaned up were sent straight for oligonucleotide-directed dideoxynucleotide chain termination sequencing reactions (HHMI Biopolymer/W). M. Keck Foundation Biotechnology Resource Laboratory, Yale University, New Haven, Conn. Those from other 16S rRNA gene PCRs were joined to a T/A plasmid vector (TOPO TA Cloning Vector for Sequencing; Invitrogen Corp. of America). ), cloned, screened by PCR, and purified (QIAGEN Miniprep DNA Purification kit).

STDs: Chlamydia

FAQ

Can fish get chlamydia?

Can chlamydia live on food?

What animal can get chlamydia?

Does tilapia carry chlamydia?

Do fish have chlamydial infections?

Chlamydial infections of fish are emerging as an important cause of disease in new and established aquaculture industries. To date, epitheliocystis, a skin and gill disease associated with infection by these obligate intracellular pathogens, has been described in over 90 fish species, including hosts from marine and fresh water environments.

What can happen if Chlamydia goes untreated?

Chlamydia is an STI caused by a specific strain of bacteria known as Chlamydia trachomatis. It is transmitted through vaginal discharge or semen. Signs of chlamydia/pain or burning while peeing/pain during sex/lower belly pain /abnormal vaginal discharge. It can cause permanent damage to a woman’s reproductive system. This can make it difficult or impossible to get pregnant later. Chlamydia can also cause a potentially fatal ectopic pregnancy/infection/infertility and pregnancy complications and chronic pain.

What is Chlamydia salmonicola?

A natural freshwater origin for two chlamydial species, Candidatus Piscichlamydia salmonis and Candidatus Clavochlamydia salmonicola, causing mixed infections in wild brown trout ( Salmo trutta)

Which Chlamydiales order causes diseases in fish?

Another described factor in the Chlamydiales order causing diseases among fish is Candidatus Similichlamydia labri, identified in gill epithelial cysts in ballan wrasse ( Labrus bergylta) in Norway (Steigen et al. 2015 ), which featured RBs, IBs and EBs.