Abstract

Over a period of 2 years, two propagating mortality epidemics, each lasting 3–4 months in duration, were observed in captive rays of a new commercial marine aquarium display in southeast Queensland. Crude mortality during the outbreaks was around 17%, and five species of rays were affected, including mangrove whip rays (Himantura granulata), estuary rays (Dasyatis fluviorum), eastern shovelnose rays (Aptychotrema rostrata), white spotted eagle rays (Aetobatus ocellatus), and blue spotted mask rays (Neotrygon kuhlii). Most of the affected rays were wild and locally caught estuary rays, originating from south-east Queensland. Sick rays showed clinical signs including anorexia, listlessness, abnormal swimming behavior or floating at the surface, whirling and cranial swelling.

Necropsies, performed on 24 rays, showed marked hepatomegaly, splenomegaly, cranial swelling and meningeal hyperemia. Histopathology revealed that all examined rays were affected with a similar assortment of lesions indicative of acute bacteraemia, including granulocytic to non-suppurative myocarditis, meningitis, and branchitis, and a variety of splenic lesions, all with intra-lesional gram-positive cocci, in pairs or chains. Cytological analysis was also performed on fluid samples aspirated from dorsal cranial swellings of two rays and showed mild granulocytic inflammation and, in one case, numerous intracellular and extracellular coccoid bacteria present in long chains. Bacterial culture was performed on swabs taken from various internal organs, including the brain, heart, kidney, spleen, liver and oviduct, of 17 rays. A catalase-negative, non-haemolytic Streptococcus sp. was isolated from 14 cases. Ten of the isolates were sent to a referral laboratory where further tests including API 20 Strep, API rapid ID 32 Strep (bioMérieux, France), and 16S rRNA PCR and sequencing were conducted on each isolate, and confirmed the identification of every isolate as S. agalactiae. Bacterial capsular serotyping initially identified the isolates as similar to molecular serotype II, with one base pair difference, when compared to reference strain EF990365 derived from the GenBank NCBI website. Further multilocus sequence typing indicated the isolates belonged to the sequence type 261 lineage, serotype 1b. This serotype was previously observed to cause an epidemic of mortality in giant Queensland grouper in northern Queensland,¹ and some of the rays of the affected display were wild-caught rays, sourced from areas where the grouper mortality had been observed. Thawed frozen fish fed to rays, and water and substrate from the ray display was consistently culture-negative during both epidemics.

Treatment with a variety of antibiotics did not alter the course of the disease, and almost all of the rays showing clinical signs subsequently died, despite in vitro sensitivity. The second outbreak ceased after autogenous vaccination of adult rays. Stocking density, intermixing of species, translocation into an artificial habitat, handling, and reduced water temperature may have been important factors predisposing to the epidemics. S. agalactiae appears to be an emerging infectious agent of wild marine fish in Australia, and streptococcosis is an important differential diagnosis in mortality epidemics of wild and captive rays, especially if accompanied by neurological signs.

Acknowledgements

The authors wish to thank other study contributors including Marnie Horton, Sea World, Dr. Mark White, Treidlia Biovet Pty Ltd, Dr. Kelly Condon, James Cook University, Dr. Fanrong Kong, Institute of Clinical Pathology and Medical Research - Westmead Hospital, Dr. Rob Jones, The Aquarium Vet, Drs. Andy Barnes and J Delamare-Deboutteville, School of Biological Sciences and Centre for Marine Science - the University of Queensland, and Dr. N. Ben Zakour, Australian Infectious Diseases Research Centre - the University of Queensland.

* Presenting author

Literature Cited

1.  Bowater RO, Forbes-Faulkner J, Anderson IG, et al. Natural outbreak of Streptococcus agalactiae (GBS) infection in wild giant Queensland grouper, Epinephelus lanceolatus (Bloch), and other wild fish in northern Queensland, Australia. Journal of Fish Diseases. 2012;35:173–186.