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Applied and Environmental Microbiology, January 2006, p. 284-290, Vol. 72, No. 1
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.1.284-290.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Molecular Evidence for Association of Chlamydiales Bacteria with Epitheliocystis in Leafy Seadragon (Phycodurus eques), Silver Perch (Bidyanus bidyanus), and Barramundi (Lates calcarifer)

Adam Meijer,^1* Paul J. M. Roholl,² Jacobus M. Ossewaarde ,^3, Brian Jones,⁴ and Barbara F. Nowak⁵

Diagnostic Laboratory for Infectious Diseases and Screening, National Institute of Public Health and the Environment, Bilthoven, The Netherlands,¹ Laboratory for Toxicology, Pathology and Genetics, National Institute of Public Health and the Environment, Bilthoven, The Netherlands,² Research Laboratory for Infectious Diseases, National Institute of Public Health and the Environment, Bilthoven, The Netherlands,³ Government of Western Australia, Department of Fisheries, Fish Health Section, South Perth, Australia,⁴ School of Aquaculture, Tasmanian Aquaculture and Fisheries Institute, University of Tasmania, Launceston, Tasmania, Australia⁵

Received 9 August 2005/ Accepted 28 September 2005

Epitheliocystis in leafy seadragon (Phycodurus eques), silver perch (Bidyanus bidyanus), and barramundi (Lates calcarifer), previously associated with chlamydial bacterial infection using ultrastructural analysis, was further investigated by using molecular and immunocytochemical methods. Morphologically, all three species showed epitheliocystis cysts in the gills, and barramundi also showed lymphocystis cysts in the skin. From gill cysts of all three species and from skin cysts of barramundi 16S rRNA gene fragments were amplified by PCR and sequenced, which clustered by phylogenetic analysis together with other chlamydia-like organisms in the order Chlamydiales in a lineage separate from the family Chlamydiaceae. By using 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. By applying immunocytochemistry, chlamydial antigens (lipopolysaccharide and/or membrane protein) were detected in gill cysts of leafy seadragon and in gill and skin cysts of barramundi, but not in gill cysts of silver perch. In conclusion, this is the first time epitheliocystis agents of leafy seadragon, silver perch and barramundi have been undoubtedly identified as belonging to bacteria of the order Chlamydiales by molecular methods. In addition, the results suggested that lymphocystis cysts, known to be caused by iridovirus infection, could be coinfected with the epitheliocystis agent.

Present address: Department of Medical Microbiology and Infectious Diseases, Erasmus Medical Center, Rotterdam, The Netherlands.