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

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. Epitheliocystis is an infection of the gills and skin of many fish species. Sometimes it can be grossly visible as cyst-like lesions, and sometimes the cysts can be seen microscopically in gill squashes, but often the only way to detect it is through histology. Epitheliocystis has been reported worldwide, both from freshwater and marine species (12). This condition is usually benign; however, sometimes it can be associated with a high mortality, particularly in cultured fish (3, 4, 26). Due to swelling of the cells of the gills and the increase in mucus around heavily infected gills, fish can become lethargic and show respiratory distress. The causative agent of epitheliocystis replicates intracellularly in the cysts and, since 1969 epitheliocystis has been associated with chlamydia-like bacteria based on the ultrastructural characteristics of the content of the cysts (4). Attempts to identify the causative agent by using monoclonal antibodies have not been successful, and their results are often inconsistent. In 1999 we discovered many new chlamydia-like sequences by using a universal Chlamydiales 16S rRNA gene PCR (30). Because of the unconfirmed ultrastructural association of epitheliocystis with chlamydia-like organisms we started investigation of archived epitheliocystis material of leafy seadragon (Phycodurus eques) and silver perch (Bidyanus bidyanus) and a new case of epitheliocystis in barramundi (Lates calcarifer) using this Chlamydiales-specific PCR. Ultrastructural analysis of epitheliocystis in these fish species was described previously, in leafy seadragon by Langdon et al. (19), in silver perch by Frances et al. (10), and in barramundi by Anderson and Prior (2). After we communicated our first preliminary positive findings during the Tenth International Symposium on Human Chlamydial Infections in Antalya, Turkey, in 2002 (25), Draghi et al. (6) undoubtedly identified a chlamydia-like bacterium as the cause of epitheliocystis in farmed Atlantic salmon (Salmo salar) using DNA sequence analysis and in situ hybridization (ISH). They proposed the name “Candidatus Piscichlamydia salmonis” for this bacterium. We describe here the characterization of the epitheliocystis agents of leafy seadragon, silver perch, and barramundi by molecular and immunocytochemical methods