Molecular evidence for association of chlamydiales bacteria with epitheliocystis in leafy seadragon (Phycodurus eques), silver perch (Bidyanus bidyanus), and barramundi (Lates calcarifer)

Abstract

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.

FIG. 1.

Phylogenetic relationships among Chlamydiales. The tree was inferred by using maximum-likelihood (ML), neighbor joining (NJ), and maximum-parsimony (MP) analysis of a 210- to 223-bp region of the 16S rRNA Chlamydiales signature sequence. The unrooted consensus tree topology is shown. Numbers indicate the percentage of times each branch appeared in a tree during 1,000 bootstrap samples (NJ and MP) or 10,000 puzzling steps (ML) in the order (NJ/MP/ML). Multifurcations connect branches for which the relative order could not unambiguously be determined. Branches supported by a bootstrap or puzzling reliability value of ≥50% with at least two treeing methods are shown. The GenBank accession numbers are given in parentheses. Included in the inference of the phylogenetic tree were (i) sequences of all type strains of classified Chlamydiales (T), (ii) all sequences of cultured chlamydia-like organisms (endosymbionts of Acanthamoeba sp. strains UWE1, TUME1, and UWC22, Parachlamydia sp. strain UWE25, Parachlamydia sp. strain UV-7, and Parachlamydia sp. strain Hall's coccus; endosymbionts of Hartmanella vermiformis and Neochlamydia hartmannellae; “Candidatus Rhabdochlamydia porcellionis”; Waddlia malaysiensis G817; “Candidatus Fritschea bemisiae”; and “Candidatus Fritschea eriococci”), (iii) cloned sequences from activated sludge that are representatives of four new environmental chlamydial lineages suggested by Horn and Wagner (15) (P-2, P-3, P-4, and P-6), (iv) cloned sequence of “Candidatus Piscichlamydia salmonis,” and (v) outgroup species Escherichia coli (GenBank accession number AE000460) and Rickettsia prowazekii (GenBank accession number M21789).

FIG. 2.

Detection of Chlamydiales bacteria in epitheliocystis of gills of barramundi (Lates calcarifer) and silver perch (Bidyanus bidyanus) by ICC and in situ RNA hybridization. (a) HE staining of gills of barramundi showing epitheliocystis cysts (arrows). (b) HE staining of lymphocystis cysts (arrows) in the skin of barramundi, clearly different in size and morphology from the cysts in the gills. (c and d) Some cysts in gills of barramundi stained clearly with antibodies raised against a membrane protein of Chlamydophila pneumoniae (c; arrow) and with antibodies raised against the lipopolysaccharide of Chlamydia trachomatis (d; arrow). (e and f) Abundant staining of cysts in gills of barramundi (e; arrows and arrowheads) and silver perch (f; arrows) by RNA ISH with a Chlamydiales-specific 16S rRNA oligonucleotide probe. Larger cysts in the gills of barramundi (e; arrowheads) stained less intensely than the smaller cysts (e; arrows). Bars: a and c to f, 10 μm; b, 100 μm.