Proteobacteria Associated with Epitheliocystis-Like Inclusions in Two Captive-Born Bonnethead Sharks (Sphyrna tiburo)
Epitheliocystis is a condition affecting gills and skin of teleost fish.1 It refers to basophilic cytoplasmic bacterial "inclusions" identified in the gills.1-4 Lesions consistent with epitheliocystis in elasmobranchs have been reported in hammerhead sharks (Sphyrna spp.), spotted eagle rays (Aetobatus narinari), spiny dogfish (Squalus acanthias), smooth dogfish (Mustelus canis), and a leopard shark (Triakis semifasciata).5-8 Lesions have been historically associated with Chlamydiales species and pan-chlamydial primers have been successfully used to obtain sequence data for a spotted eagle ray and a leopard shark.6,8,9 Recently, advanced diagnostic techniques and molecular analysis have suggested the lesions are actually associated with proteobacteria.2,3 Inclusions morphologically similar to epitheliocystis were found in gills of two captive-born, wild-conceived, bonnethead shark (Sphyrna tiburo) pups. Histopathology of gills revealed typical spherical, granular, basophilic inclusions containing fine filamentous bacterial rods within epithelial cells arising from the lamellae. The organisms are gram negative, and highlighted with Giemsa and Macchiavello stains, but do not stain with periodic acid-Schiff. Immunohistochemistry was performed using monoclonal mouse and polyclonal rabbit anti-chlamydial antibodies, showing strong positive staining with the polyclonal antibody, but not with the monoclonal product. Polymerase chain reactions (PCR) and Sanger sequencing were performed on samples from one pup. Initial PCR with pan-Chlamydiales 16S rRNA gene primers and resultant molecular sequence data support findings that epitheliocystis could be caused by unclassified proteobacteria species. Two adult, wild-caught bonnethead sharks screened for epitheliocystis-like inclusions revealed a similar inclusion indicating that this condition occurs in free-ranging populations.
The authors thank Heather Broadhurst, Hap Fatzinger, Kent Passingham, Emily Peele, and Clint Taylor for their support of this project.
* Presenting author
+ Student presenter
1. Nowak BF, LaPatra SE. Epitheliocystis in fish. J Fish Dis. 2006;29(10):573–588.
2. Contador E, Methner P, Ryerse I, Huber P, Lillie BN, Frasca S, Lumsden JS. Epitheliocystis in lake trout Salvelinus namaycush (Walbaum) is associated with a β-proteobacteria. J Fish Dis. 2015. In press.
3. Toenshoff ER, Kvellestad A, Mitchell SO, Steinum T, Falk K, Colquhoun DJ, Horn M. A novel betaproteobacterial agent of gill epitheliocystis in seawater farmed Atlantic salmon (Salmo salar). PLoS One. 2012;7(3):E32696.
4. Mitchell SO, Steinum TM, Toenshoff ER, Kvellestad A, Falk K, Horn M, Colquhoun DJ. 'Candidatus Branchiomonas cysticola' is a common agent of epitheliocysts in seawater-farmed Atlantic salmon Salmo salar in Norway and Ireland. Dis Aquat Org. 2013;103(1):35–43.
5. Garner MM. A retrospective study of disease in elasmobranchs. Vet Pathol. 2013;50(3):377–389.
6. Camus A, Soto E, Berliner A, Clauss T, Sanchez S. Epitheliocystis hyperinfection in captive spotted eagle rays Aetobatus narinari associated with a novel Chlamydiales 16S rDNA signature sequence. Dis Aquat Org. 2013;104(1):13–21.
7. Borucinska JD, Frasca S. Naturally occurring lesions and micro-organisms in two species of free-living sharks: the spiny dogfish, Squalus acanthias L., and the smooth dogfish, Mustelus canis (Mitchill), from the north-western Atlantic. J Fish Dis. 2002;25(5):287–298.
8. Polkinghorne A, Schmidt-Posthaus H, Meijer A, Lehner A, Vaughan L. Novel Chlamydiales associated with epitheliocystis in a leopard shark Triakis semifasciata. Dis Aquat Org. 2010;91(1):75–81.
9. Corsaro D, Greub G. Pathogenic potential of novel Chlamydiae and diagnostic approaches to infections due to these obligate intracellular bacteria. Clin Microbiol Rev. 2006;19(2):283–297.