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.

Molecular Characterization of “Candidatus Similichlamydia latridicola” gen. nov., sp. nov. (Chlamydiales: “Candidatus Parilichlamydiaceae”), a Novel Chlamydia-Like Epitheliocystis Agent in the Striped Trumpeter, Latris lineata (Forster)

“…Detection of the Chlamydia-like organism within the epitheliocystis cysts by ISH in 5-m serial sections was conducted with Chlamydiales-specific antisense (digoxigenin [DIG]-ATG TG[T/C] TAC TAA CCC TTC CGC CAC TA-DIG) and sense (DIG-ATC CTA CGC TAC TAA GTC TCT CAT CA-DIG) DIG-labeled oligonucleotide probes as described previously (12,20), with some modifications. Briefly, the sections were dewaxed, washed with phosphate-buffered saline (PBS) for 3 min, and digested for 30 min at 37°C in 5 mg/liter proteinase K (Sigma) in 0.1 mol/liter Tris-HCl (pH 7.6).…”

“…The latter disease is a condition of the skin and gills and is generally associated with infections by Chlamydia-like organisms (CLOs) (7)(8)(9)(10). These CLOs are Gram-negative, intracellular bacteria that may cause cyst-like lesions in the gill lamellae (9,11,12). The lesions may lead to epithelial hyperplasia and inflammation of the infected tissues, increased mucus production, and respiratory distress, sometimes ending in death (12)(13)(14)(15).…”

“…These CLOs are Gram-negative, intracellular bacteria that may cause cyst-like lesions in the gill lamellae (9,11,12). The lesions may lead to epithelial hyperplasia and inflammation of the infected tissues, increased mucus production, and respiratory distress, sometimes ending in death (12)(13)(14)(15). Most reported losses in aquaculture attributed to epitheliocystis occur during the larval or juvenile culture stage (16).…”

Molecular Characterization of “Candidatus Parilichlamydia carangidicola,” a Novel Chlamydia-Like Epitheliocystis Agent in Yellowtail Kingfish, Seriola lalandi (Valenciennes), and the Proposal of a New Family, “Candidatus Parilichlamydiaceae” fam. nov. (Order Chlamydiales)

“…Although epitheliocystis is often benign, it can cause epithelial hyperplasia and inflammation of infected tissues, resulting in significantly reduced growth (12,13). In severe cases of hyperinfection, the cyst-like branchial lamellar lesions can result in increased lamellar fusion with consequent respiratory distress and death (14,15). Epitheliocystis outbreaks have been reported in many aquaculture species, including barramundi, Lates calcarifer (16); white sturgeon, Acipenser transmontanus (17); silver perch, Bidyanus bidyanus (18); Atlantic salmon, Salmo salar (19); red sea bream, Pagrus major (20); carp, Cyprinus carpio (21); and yellowtail kingfish, S. lalandi (6).…”

Pathologic Findings in Weedy (Phyllopteryx taeniolatus) and Leafy (Phycodurus eques) Seadragons

“…Epitheliocystis has previously been reported in the leafy seadragon, 13 with molecular evidence associating Chlamydiales bacteria with the infection. 17 By far the most serious infectious diseases affecting WSD and LSD are scuticociliatosis, mycobacteriosis, and infection by melanized fungi (eg, Exophiala spp). Strategies for improving captive longevity of this species will need to concentrate on prevention and control of these organisms.…”