Chlamydial classification

Piscichlamydia and other chlamydia-like agents in epitheliocystis diseases of fish

Introduction

Chlamydia-like agents in fish were originally recognised by Hoffman et al., 1969, with the first molecular characterisation in 2002. A new species associated with epitheliocystis in farmed Atlantic salmon, Piscichlamydia salmonis, was proposed in 2004, prompting the article by Mathias Horn with its emphasis on potential evolutionary significance, which appears below. Subsequently it has become clear that the chlamydia-like agents associated with epitheliocystis are remarkably heterogeneous and a further Candidatus species, Clavochlamydia salmonicola was described in 2008. Here the original article of Mathias Horn is retained unaltered and a brief update by myself follows.

[MEW] January 2008

“Candidatus Piscichlamydia salmonis” - getting closer to the root of chlamydiae.

“Candidatus Piscichlamydia salmonis” represents a novel family within the phylum Chlamydiae and is the deepest branching "chlamydia-like" organism identified so far. Analysis of these bacteria, which cause epitheliocystis in salmon but which have so far not been obtained in cell culture, might reveal novel insights into the evolution of chlamydiae".

Epitheliocystis has been associated with heavy mortality of farmed fish and reduced growth in the survivors. Histologically the disease is characterized by the presence of intracytoplasmic, granular inclusions in branchial epithelial cells. Bacteria with a "chlamydia-like" morphology have been observed in epitheliocystis lesions many years ago [Langdon et al., 1991]. Meijer and Ossewarde, 2002 presented the first molecular evidence for the presence of chlamydiae in the gill lesions of epitheliocystis in a paper at the Tenth International Symposium on Human Chlamydial Infections. Frasca and co-workers have now identified chlamydiae in the gill lesions of farmed Atlantic salmon (from Norway and Ireland, respectively) by 16S rRNA gene sequencing, electron microscopy and _ in situ_ hybridization [Draghi et al., 2004]. These chlamydiae formed large inclusions containing elongated, oblong, or spherical reticulate bodies (0.7-1.8 Ám) and round to oval intermediate bodies (0.6-0.8 Ám). Elementary bodies could only be observed in the specimens from Irish salmon which also contained a fourth morphological form, a "head-and-tail" cell with long unipolar extensions with terminal knobs and central condensed nucleoids. Riboprobes transcribed from cloned near-full-length 16S rDNA amplicons from Norwegian gills hybridized with inclusions in proliferative lesions from Irish and Norwegian fish. Comparative sequence analysis of the 16S rRNA genes amplified from these samples showed that the chlamydiae in Norwegian and Irish salmon were almost identical (99% sequence similarity). However, they were only distantly related to known chlamydiae (> 80% sequence similarity) and thus represent a novel genus of a novel family within the order Chlamydiales. In view of the salmon host and in accordance with taxonomic practice, the authors suggested the name “Candidatus Piscichlamydia salmonis_” for designation of these bacteria, the term Candidatus indicating that the organism has not (yet) been cultured _ in vitro.

Interestingly, “Candidatus Piscichlamydia salmonis” did not show particularly high 16S rRNA sequence similarity with the partial, chlamydia-like, 16S rRNA gene sequences obtained previously by Ossewaarde from silver perch, Barramundi cod, and leafy sea dragon (these were more similar to other environmental chlamydiae belonging to the family Parachlamydiaceae). Computer-based phylogenetic analysis demonstrated that “Candidatus Piscichlamydia salmonis” does not group with previously described chlamydiae but forms the deepest branch yet observed within the phylum Chlamydiae (see figure 1).

Phyllogenetic relationships of Piscichlamydia

Figure 1. [Double click on thumbnail]. Phylogenetic consensus tree (based on TREEPUZZLE, neighbour joining, and maximum parsimony analysis) showing the affiliations of described members of the phylum Chlamydiae with their closest relatives (Verrucomicrobia and the “vadin” clones). Bar, 10% estimated evolutionary distance. (A. Collingro is acknowledged for phylogenetic analysis.)

Surprisingly Draghi et al., 2004 detected binding of monoclonal LPS antibodies by immunogold labelling, suggesting the presence of the alpha-KDO-(2-->8)-alpha-KDO-(2-->4)-alpha-KDO epitope thought to be a unique character of members of the Chlamydiaceae. The authors suggest this might also be explained by cross reactivity of the anti-LPS antibodies with a related but as yet uncharacterized trisaccharide.

No efforts have yet been made to cultivate “Candidatus Piscichlamydia salmonis” in fish cell lines. It is tempting to speculate that “Candidatus Piscichlamydia salmonis_” is able to grow in free-living amoebae since all chlamydiae either naturally thrive in these protozoa or are able to use them as alternative hosts. The findings reported by Draghi _et al., 2004 extend knowledge on the diversity and distribution of chlamydiae as well as throwing new insight on a commercially important disease of fish. Their findings are also fascinating from an evolutionary point of view. As the deepest branching chlamydia, a more detailed analysis of “Candidatus Piscichlamydia salmonis” should reveal novel insights into the evolution of chlamydiae and might allow further conclusions on the last common ancestor of all chlamydiae.

Mathias Horn, November 2004


Update January 2008.

Epitheliocystis affects both the gill and skin epithelia in more than 50 species of freshwater or marine fish, including non teleost fish such as the white sturgeon and leopard shark. It is commonest in artificially cultured fish and has been reported particularly problematic in cultured beam and bass [Kent et al., 2002]. The disease is thought to be caused by intracellular infection with gram negative, non cultivable bacteria. Progressive enlargement of infected epithelial cells results in unicellular cysts (hence epitheliocystis) in the skin or gills containing If fish are affected in the gills, the inflammatory response of the host may become severe and in some cases this could lead to respiratory problems. This condition is commonly termed proliferative gill inflammation (PGI). The cysts contain fine basophilic granular inclusions. Electron microscopy shows within the inclusions either the classic bimorphic chlamydial developmental cycle or an atypical developmental cycle with primary and intermediate long cells. Similar atypical forms are shown by _Rhabdochlamydia. Epitheliocystis and PGI are generally benign, though in severe cases mortalities approaching 100% have been reported - for a detailed review see Nowak & LaPatra, 2006. Where necessary the condition can be successfully treated with tetracycline or chloramphenicol. Paramyxoviruses have also been reported to be associated with epitheliocystis, but not consistently.

Molecular characterization of chlamydia-like agents in epitheliocystis lesions from 5 different species of fish have been performed in several studies. The associated fish species were: leafy seadragon ( Phycodurus eques), silver perch ( Bidyanus bidyanus), and barramundi ( Lates calcarier) [Meijer et al., 2006]; the Atlantic salmon ( Salmo salar) [Draghi et al., 2004] and the Arctic charr ( Salvelinus alpinus) [Draghi et al., 2007]. 16S rRNA gene fragments form gill cysts from the first three fish species and from skin cysts of barramundi were amplified by PCR then sequenced. By in situ RNA hybridization, 16S rRNA Chlamydiales -specific sequences were detected in gill cysts of silver perch and in gill and skin cysts of barramundi. Immunocytochemistry demonstrated chlamydial antigens (lipopolysaccharide and/or membrane protein) in gill cysts of leafy seadragon and in gill and skin cysts of barramundi, but not in gill cysts of silver perch. Phylogenetic analyses showed the nucleotide sequences clustered together with other chlamydia-like organisms in the order Chlamydiales. The phylogenetic lineage and ultrastructure of these three agents was quite distinct from the family Chlamydiaceae and from each other [Meijer et al., 2006]. They were also quite distinct from the chlamydia-like agent Piscichlamydia salmonis which was associated with epitheliocystis in Atlantic salmon [Draghi et al., 2004].

Subsequently chlamydial-like agents in epitheliocystis material from the Arctic charr were identified consisting of membrane bound inclusions containing round to elongate reticulate bodies which were immunoreactive with antibody to chlamydial lipopolysaccharide. This material yielded 16srRNA amplification products which were closely related to Neochlamydia hartmanellae [Draghi et al., 2007]. This is the fourth Neochlamydia spp sequence to be associated with epitheliocystis. Taken together these results suggest that a variety of heterogeneous chlamydia-like agents, not just Piscichlamydia salmonis are associated with epitheliocystis in fish. The presence of a clinical neochlamydial sequence, first identified from a cat, in Arctic charr suggests a possible mammalian and piscine host range for some environmental chlamydiae. However in epitheliocystis in most fish species, reactivity with antibody to chlamydial lipopolysachharide is negative [Nowak & LaPatra, 2006] indicating either that not all cases are caused by chlamydia-like agents or that there may be differential expression of lipopolysaccharide antigen during the developmental cycle. The high diversity and host specificity of the epitheliocystis agents indicates that there is only limited transmission between fish species.


Figures 2 - 6. Epitheliocystis in Barramundi and Silver Perch. Figures 2 - 6 provided by courtesy of Adam Meijer, Siska Gielis-Proper and Paul Roholl, National Institute for Public Health and the Environment, Bilthoven, the Netherlands. Copyright Adam Meijer.

ISH 16S rRNA epitheliocystis in barramundi

higher magnification

Epitheliocystis in barramundi

Fig. 2. Abundant staining of epitheliocystis cysts in gills of barramundi ( Lates calcarifer) by RNA insitu hybridisation (ISH) with a Chlamydiales -specific 16S rRNA oligonucleotide probe. Double click to enlarge the thumbnails. Fig. 3. Larger magnification of epitheliocystis cysts in gills of barramundi ( Lates calcarifer) stained by RNA ISH with a Chlamydiales-specific 16S rRNA oligonucleotide probe. Fig 4. Epitheliocystis cyst in gills of barramundi ( Lates calcarifer) stained by monoclonal antibody CF-2 directed against Chlamydia trachomatis LPS.

Cysts in barramundi stained with MAb to LPS

ISH 16srRNA Silver perch

Fig 5. Epitheliocystis cyst in gills of barramundi ( Lates calcarifer) stained by monoclonal antibody RR-402 directed against Chlamydophila pneumoniae membrane protein. Larger cysts stained less intensely than smaller cysts, possibly due to reduced amounts of RNA in EB compared to RB and the loss of cross-reactive epitopes during maturation of LPS or proteins. Fig 6. Epitheliocystis cysts in gills of silver perch ( Bidyanus bidyanus) stained by RNA ISH with a Chlamydiales-specific 16S rRNA oligonucleotide probe.

For further details see Meijer et al., 2006.


Figures 7 - 10. Epitheliocystis in Red Snapper. Figures 7 - 10 by kind permission of Dr Brian Jones, Dept of Aquaculture and Animal Health, Western Australia. Copyright Brian Jones.

Cysts in red snapper, h & e

higher mag picture

TEM cysts in red snapper

higher mag TEM

Fig 7. H and E stained paraffin section of gills of Red Snapper ( Pagrus auratus) showing cysts due to Epitheliocystis. Original photomicrograph 100x. Fig 8. H and E stained paraffin section of gills of Red Snapper ( Pagrus auratus) showing detail of a cyst. Original photomicrograph 400x. Fig 9. Transmission electron micrograph of a portion of a cyst from Red Snapper. Note structures characteristic of the developmental cycle of the chlamydiae. Original photomicrograph 3,900x. Fig 10. As Fig 9, showing detail of chlamydia-like reticulate bodies within a cyst. Original photomicrograph 39,000x.

[PICK MEW comment: It is unclear whether chlamydia-like bacteria are responsible for all cases of salmon epitheliocystis; a paramyxovirus has also been associated with the disease [Fridell et al., 2004]. Epitheliocystis is considered particularly problematic in cultured bream and bass [Kent et al., 2002] in which chlamydia-like agents might also occur].

Clavochlamydia salmonicola

Using 16SrRNA sequencing and in situ hybridisation, Karlsen et al., 2008 identified a new Candidatus chlamydia species, different to Piscichlamydia, from the gills of Atlantic salmon and trout in freshwater in Norway. The bacterial agent showed morphologically different developmental stages within the vacuole of infected epithelial cells consisting. Reticulate bodies consisted of up to 2 μm large spherical or elongated, branching bodies containing ribosomes and chromatin. Chromatin rich intermediate bodies were between 350 and 650 nm in diameter while the electron dense elementary bodies were approximately 1 μm long and consisted of a head region (about 350–400 nm in diameter) and a 550–600 nm long tail region. Phyllogenetic analysis indicates that the new organism is loosely affiliated to the family Chlamydiaceae _and may have branched from the rest of the _ Chlamydiaceae prior to the Chlamydia - Chlamydophila split. The organism was called Candidatus Chlavochlamydia salmonicola. The authors suggest that the epitheliocystis agent is not one bacterium, but a genetically diverse group of intracellular parasites only distantly related within the Chlamydiales. It is possible that different chlamydiae inhabit different aquatic habitats, eg salt water versus fresh water.

See also: _ Classification.ParaChlamydiaceae_ Environmental chlamydia Chlamydiales diversity Rhabdochlamydia_

[MEW] March 14th 2008

References

Draghi, A. 2nd., Popov, V. L., Kahl, M. M., Stanton, J. B., Brown, C. C., Tsongalis, G. J., West, A. B. & Frasca, S. Jr. (2004). Characterization of "Candidatus Piscichlamydia salmonis" (order Chlamydiales), a chlamydia-like bacterium associated with epitheliocystis in farmed atlantic salmon (Salmo salar). Journal of Clinical Microbiology 42, 5286 - 5297

Draghi, A. 2nd., Bebak J., Popov, V. L., Noble, A. C., Geary, S. J., West, A. B., Byrne, P. & Frasca, S Jr. (2007). Characterization of a Neochlamydia-like bacterium associated with epitheliocystis in cultured Arctic charr Salvelinus alpinus. Diseases of aquatic organisms 76 (1), 27 - 38.

Fridell, F., Devold, M. & Nylund, A. (2004). Phylogenetic position of a paramyxovirus from Atlantic salmon Salmo salar. Diseases of aquatic organisms 59, 11-15 (2004). Full paper

Hoffman, G.L., Dunbar, C.E., Wolf, K., and Zwillenberg, L.O. (1969) Epitheliocystis, a new infectious disease of the bluegill (Lepomis macrochirus). Antonie Van Leeuwenhoek 35: 146–158.

Karlsen, M., Nylund, A., Watanabe, K., Helvik, J. V., Nylund, S. & Plarre, H. (2008). characterization of 'Candidatus Clavochlamydia salmonicola': an intracellular bacterium infecting salmonid fish. Environmental Microbiology 10 (1), 208 - 218. Full paper

Langdon, J. S., Elliott, K., MacKay, B. (1991). Epitheliocystis in the leafy sea-dragon. Australian Veterinary Journal 68, 244.

Meijer A, & Ossewaarde, J. M. (2002). Description of a wider diversity within the order Chlamydiales than currently classified. pp 13-16, in Chlamydial Infections. Proceedings of the tenth international symposium on human chlamydial infections. Schachter J et al. (eds.) International Chlamydia Symposium: San Francisco, USA.

Meijer, A., Roholl, P. J., Ossewaarde, J. M., Jones, B. & Nowak, B. F. (2006) Molecular evidence for association of Chlamydiales bacteria with epitheliocystis in leafy seadragon (Phycodurus eques), silver perch (Bidyanus bidyanus), and barramundi (Lates calcarier). Applied and Environmental Microbiology 72 (1) 284 - 290. Full paper

Nowak, B. F. & LaPatra, S. E. (2006). Epitheliocystis in fish. Journal of Fish Diseases 29 (10), 573 - 588. [PICK A useful review].

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Topic revision: r3 - 2010-08-27 - MeWard
 
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