Epizootics of Streptococcus Agalactiae Infection in Captive Rays from Queensland, Australia

DR RACHEL OLIVE BOWATER (Orcid ID : 0000-0003-2823-1284)

Article type : Original Manuscript

Epizootics of Streptococcus agalactiae infection in captive rays from Queensland, Australia

R O Bowater,1* M M Dennis,2 D Blyde,3 B Stone,4 A C Barnes, 5 J Delamare-Deboutteville,5 M A Horton,3 M White,6 K Condon,7 R Jones8

1Biosecurity Queensland, Department of Agriculture & Fisheries, PO Box 1085. Townsville, Queensland, Australia, 4810. [email protected]

2Ross University School of Veterinary Medicine, PO Box 334, Basseterre, St. Kitts and Nevis.

3Sea World. PO Box 190, Surfers Paradise, Queensland, Australia. 4217.

4QML Vetnostics, 11 Riverview Place, Metroplex on Gateway, Murrarie, Queensland, Australia.

5 School of Biological Sciences and Centre for Marine Science, The University of Queensland, Brisbane, Qld, Australia.

6Treidlia Biovet Pty Ltd, Unit 76 Powers Business Park, 45 Powers Rd Seven Hills, PO Box 6563 Seven Hills, New South Wales, Australia. 2147.

7James Cook University, School of Veterinary and Biomedical Sciences, Townsville, Queensland, Australia. 4811.

8The Aquarium Vet. PO Box 2327 Moorabbin, Victoria, Australia. 3189. Author Manuscript

This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/jfd.12701

ABSTRACT

The aim of this paper is to describe two epizootics of high mortalities from infection with Streptococcus agalactiae, occurring in captive rays held in a marine display aquarium in south east Queensland, Australia in 2009 and 2010. Five different species of rays were affected, including mangrove whiprays (Himantura granulata), estuary rays (Dasyatis fluviorum), eastern shovelnose rays (Aptychotrema rostrata), white spotted eagle rays (Aetobatus narinari), and blue spotted mask rays (Neotrygon kuhlii). This report describes the history of both epizootics including collection, quarantine and husbandry of rays, the disease epizootics, clinic-pathological features of the disease, antimicrobial therapy, autogenous vaccine production, and laboratory studies including clinical and histopathology, bacteriology, PCR, molecular serotyping and sequencing of the bacterium Streptococcus agalactiae.

Key words streptococcosis; bacterium; ray; elasmobranch, Streptococcus agalactiae; fish

INTRODUCTION Streptococcus agalactiae is a significant bacterial pathogen of animals, largely known for causing mastitis in cows, but also causing a variety of conditions such as meningoencephalitis, necrotizing fasciitis, pyometra, and generalised septicaemia in many terrestrial animals including horses, camels, dogs, cats, monkeys, hamsters, mice, rabbits, and lizards (Edelsten & Pegram 1974; Elliott, Facklam & Richter 1990; Lämmler et al. 1998; Yildirim, Lämmler & Weiss 2002; Hetzel et al 2003; Memarium 2009; Ren et al. 2014). In humans, S. agalactiae is a cause of morbidity in neonates and can cause septicaemia and meningitis in immuno-compromised elderly people (Edwards & Baker 2005; Harris et al. 2011). In aquatic animals, S. agalactiae has been reported to cause septicaemia and meningitis in farmed and wild fish, necrotizing fasciitis and septicaemia in captive reared juvenile saltwater crocodiles and dolphins, and necrotizing splenitis, hepatitis and renal haemorrhage in frogs. (Plumb et al. 1974; Amborski et al. 1983; Direkburakum & Danayadol 1987; Baya et al. Author Manuscript 1990; Evans et al. 2002; Duremdez et al. 2004; Zappulli et al. 2005; Evans, Klesius & Shoemaker 2006a; Evans et al. 2006b; Bishop et al. 2007; Bowater et al. 2012).

Streptococcosis is a significant disease affecting cultured and wild fish worldwide, and caused by a variety of streptococcal bacteria including Streptococcus agalactiae, S. dysgalactiae, S. ictaluri, S. iniae, S. phocae, S. parauberi and S. uberis (Evans et al. 2006a; Romalde et al. 2008; Camus et al. 2008; Anshary et al. 2014). Most reported epizootics of S. agalactiae causing widespread mortalities of cultured fish have occurred in purebred and hybrid tilapia species, both in hatcheries and on grow-out farms in countries including Brazil, China, Columbia, Indonesia, Israel, Malaysia, Thailand, and the United States of America (USA) (Shoemaker, Evans & Klesius 2000; Hernández, Figueroa & Iregui 2009; Mian et al. 2009; Musa et al. 2009; Amal et al. 2013; Huang et al. 2013; Anshary et al. 2014; Lusiastuti et al. 2014). Associated aquaculture losses from S. agalactiae have been estimated at US$10 million in the USA with US$100 million globally. S. agalactiae epizootics have also been reported in other cultured fish species including ‘ya-fish’ (Schizothorax prenanti) cultured in earthen ponds in China, in seabream (Sparus auratus) from Kuwait, silver pomfret (Pampus argenteus) used in foot spa pedicures, golden pomfret/ pompano (Trachinotus blochii) cage- cultured in Malaysia and Singapore, and barcoo grunter (Scortum barcoo) cultured in China (Evans et al. 2002; Duremdez et al. 2004; Amal et al. 2012; Geng et al. 2012; Liu et al. 2014; Chong, Wong & Lee et al. 2017).

In contrast, wild fish kills involving S. agalactiae often involve a variety of marine fish and occasionally elasmobranchs, including mullet (Liza klunzinger), bluefish (Pomatonus saltatrix), striped bass (Morone saxatilis) and sea trout (Cynoscion regalis), Queensland grouper (Epinephelus lanceolatus) from north-eastern Australia, and have been reported worldwide from the USA, Kuwait, Israel and Australia (Plumb et al. 1974; Baya et al. 1990; Evans et al. 2002; Evans et al. 2006a; Bowater et al. 2012; Bowater 2015). This is the first report of streptococcosis from Streptococcus agalactiae involving wild and captive rays.

MATERIALS AND METHODS Ray collection and source Rays were collected from the wild specifically for the purpose of establishing an exhibit pool at a marine aquarium. No marine animals were in the exhibit prior to this occurring. The Author Manuscript initial population of rays consisted of both mature and juvenile rays, sourced locally, from marine waters extending from the Gold Coast to Moreton Bay in south east Queensland, from October 2008 through to March 2009. These rays were caught using seine nets and were transported back to the aquarium in oxygenated tubs. Wild-caught rays consisted of a variety

of different species including; estuary (or brown) rays Dasyatis fluviorum (Ogilby 1908), blue spotted fantail rays Taeniura lymma (Forsskal 1775), giant shovelnose rays Glaucostegus typus (Bennet 1830), white spotted eagle rays Aetobatus narinari (Euphrasen, 1790), blue spotted mask rays Neotrygon kuhlii (Müller & Henle 1841), shovelnose rays Aptychotrema rostrata (Shaw 1794) and whiprays Himantura toshi (Whitley 1939).

Eleven rays were sourced from a licensed fish collector in north Queensland, from waters in far north-eastern Queensland. These rays included; two eastern fiddler rays Trygonorrhina fasciata (Müller & Henle 1841), that were long term residents; a mangrove whipray Himantura granulata (Macleay 1883) purchased in October 2008, quarantined, then transferred to the ‘exhibit pool’ in January 2009; and eight adolescent blue spotted fantail rays (Taeniura lymma) purchased later in 2009 quarantined, and introduced into the ‘exhibit pool’ in mid-June 2009. A small number of wild caught rays, that were too young to tag (wing width < 28 cm), and ray pups subsequently born in captivity in the exhibit pool, were placed in a separate, adjoining, ‘baby pool’ for several weeks, then later transferred back into to the main pool.

Animal husbandry The new ‘exhibit pool’ had a volume capacity of 434,000 L and this amount of sea water was fully exchanged via pump every 72 min. Before being introduced into the exhibit pool, each ray was tagged with a passive integrated transponder (PIT) tag and immersed for in an antiparasitic bath containing Neguvon® (Bayer Australia Ltd.) at a final concentration of 0.25 ppm. Each animal had its barb clipped and its wing width measured. Rays were fed daily with a mixture of thawed frozen fish including pilchards Sardinops sagax (Jenyns 1842), whiting Sillago ciliata (Cuvier 1829), Pacific saury Cololabis saira (Brevoort 1856), squid Sepioteuthis australis (Quoy & Gaimard 1832), New Zealand green-lipped mussels Perna canaliculus (Gmelin 1791) and eastern king prawns Penaeus (melicertus) plebejus (Hess 1865). Rays were under constant observation during daylight hours and those found dead had their PIT tag number and dates of death recorded, before being examined post-mortem and necropsied. Author Manuscript

Necropsy and histology Necropsies were performed on eleven sick rays in 2009 (of which 10 were sampled for histopathology), and on sixteen sick rays in 2010 (of which 14 were sampled for

histopathology). Gill, brain, heart, kidney, spleen, liver, gonad and intestine were sampled variously from each ray, immersed in 10% formalin in sea water for 24 to 48 h and processed routinely for histopathology using standard methods. Tissues containing bony elements, such as skin or gill, were decalcified where necessary by immersing in 5% nitric acid for 30 min prior to processing. Sections were cut at 5 μm, stained with haematoxylin and eosin (H&E) and, where indicated by pathological findings, re-cut tissues were stained with Brown-Hopps Gram stain.

Bacteriology Bacterial culture was performed on swabs taken from various internal organs, including the brain, heart, kidney, spleen, liver and oviduct, from six rays in 2009 and eleven rays in 2010. Bacteriology was initially undertaken at a commercial veterinary pathology laboratory. Ten of these isolates were sent to a referral Queensland Government Veterinary Laboratory in Townsville, Queensland. Swabs were streaked onto 5% sheep blood agar and incubated at 25oC for 48 to 72 h. Further tests including API 20 Strep, API rapid ID 32 Strep (bioMérieux, France), and PCR were conducted on each isolate for bacterial identification to species.

16S PCR and sequencing

Amplification and sequencing of the 16S rRNA region was performed on five S. agalactiae isolates. High molecular weight genomic bacterial DNA (gDNA) was extracted (as outlined in Bowater, 2015). Positive amplicons of 1300bp were visualised by agarose gel electrophoresis. PCR amplicons were purified using QIAquick PCR purification Kit (Qiagen, Australia), eluted in 50 µl water and sequenced using the 63F or 1387R primer (2 pmol µL-1) and BigDye Terminator v 3.1 Cycle sequencing Kit (Applied Biosystems, USA). Sequences were edited using Sequencher 4.0 (Gene Codes Corporation, USA) and compared to sequences on GenBank using the BLASTn algorithm.

Bacterial molecular serotyping and multilocus sequence typing The concern for zoonotic potential during the disease outbreaks at a commercial marine

display exhibit aquariumAuthor Manuscript manned by numerous staff, prompted molecular studies to be done on the bacterial isolates, to determine the molecular serotype and whether the ray isolates were related to human isolates. Five S. agalactiae isolates; including three isolates from each of three estuary rays (Dasyatis fluviorum), one isolate from a mangrove whipray (Himantura

granulata), and one isolate from an eastern shovelnose ray (Aptychotrema rostrata) were serotyped, using two molecular serotyping methods for epidemiological tracing; multilocus sequencing typing and molecular serotyping. These were derived from whole genome sequencing, as previously described (Delamare-Deboutteville 2014; Bowater 2015). Briefly, genomic fragment libraries for whole genome sequencing of the five ray isolates were prepared at the Australian Genome Research Facility (AGRF) using Nextera paired-end DNA library preparation protocol. Libraries were pooled for sequencing on the Illumina HiSeq 2000 instrument at the AGRF according to manufacturer’s protocols (refer to Delamare- Deboutteville 2014; Bowater 2015). After quality trimming, draft assemblies of the 100 bp PE reads were conducted with Velvet. The whole draft genome sequence of each of the ray isolate was submitted directly to the MLST database website, where a BLAST search was performed to determine each individual allelic profile and sequence type (Bentley et al. 2006). A complete sequence of the cps operon of each ray isolate was derived from associated draft genome sequence. Reference sequences for the ten serotypes published in the literature (Ia, Ib, II to IX) were retrieved from the GenBank database on the NCBI website (Table I), and BLASTn comparisons were performed to ascertain/confirm the serotype of each isolate (Delamare-Deboutteville, 2014).

Clinical pathology Cytological analysis was also performed on subcutaneous fluid samples aspirated from dorsal cranial swellings of two rays. Aspirated fluid was submitted in an EDTA-containing haematological tube. Cytological evaluation followed preparation of a direct and a cytospin slide using 500 µL of the EDTA-containing fluid using a Shandon Cytospin III centrifuge (Thermo Fisher Scientific, Scoresby, VIC, Australia) at 1,200 RPM for 5 min with the slides then stained with Quick Dip (Point of Care Diagnostics, Artarmon, NSW, Australia) as per manufacturer’s directions.

Autogenous vaccination Following the initial diagnosis of Streptococcus agalactiae in May 2009, an isolate was Author Manuscript forwarded to Mark White at Treidlia Biovet Pty Ltd, and an autogenous vaccine was manufactured. The isolate was initially stored as a seed culture in glycerol in liquid nitrogen, then rejuvenated onto sheep blood agar plates and incubated overnight at 25oC. Several colonies were inoculated into 20 mL Tryptic Soy Broth (Oxoid Microbiology Products,

Thermo Scientific) and incubated overnight at 25oC. The starter culture was inoculated into 1.0 L Tryptic Soy Broth and incubated for a further 18 h at 25oC with agitation. Bacterial cultures were inactivated with 0.3% formalin. The bulk antigen was tested for purity, bacterial count and complete inactivation. After adjustment of the bulk antigen to 1 x 109 cfu mL-1, Montanide ISA 206 adjuvant (Seppic) was added at a ratio of 1:1. Mixing was performed in an IKA laboratory mixer twice at 2,000 RPM for 10 minutes to form a double emulsion. The finished vaccine was tested for sterility, formalin level and stability of emulsion. On 19 November 2009, all rays were vaccinated intra-peritoneally with either 1 mL vaccine (animals less than 5 kg bodyweight) or 2 mL (animals greater than 5 kg bodyweight), initially at a four week interval and then annually. RESULTS Epizootics Once the exhibit pool had been populated, an occasional mortality of ‘unknown’ cause was observed sporadically until March 2009, when 13 rays were found dead. The presumptive diagnosis for this initial mortality cluster was ‘gas bubble disease’. Although not confirmed, rays were subsequently removed from the exhibit pool for ten days, during which time, one ray died. All rays were then transferred back into the ‘exhibit pool’.

The first epizootic began in May 2009, with clusters of four to five deaths occurring in a ‘cyclical’ pattern. Sick rays showed clinical signs including anorexia, listlessness, abnormal swimming behaviour or floating at the surface, whirling and cranial swelling. This epizootic included mainly southern ray species, and a single mangrove whipray (Himantura granulata) (sourced from northern Queensland) that died in June 2009. The first epizootic ceased in late July 2009. Pathology done on deceased rays returned the same diagnosis, indicative of bacterial septicaemia due to Streptococcus agalactiae. The results are summarised in Table II. Two further sporadic deaths, recorded as ‘unknown’ occurred between both epizootics.

The second epizootic commenced in July 2010, with the death of a recently introduced estuary ray, and a vaccinated animal, both dying of laboratory-confirmed S. agalactiae infection. A propagating epidemic followed where a total of 30 rays died by mid-October. Author Manuscript Dead rays from this epizootic had clinical pathology and/or laboratory confirmed evidence of infection with S. agalactiae. After four deaths, all adult rays were vaccinated with an autogenous vaccine. The second epizootic ceased in October 2010.

Biosecurity The exhibit was closed to the public during both epizootics. Staff awareness was raised regarding potential zoonosis, as the bacterium serotype was unknown at the time. Staff wore personal protective gear and carried out good personal hygiene. All diving in the exhibit aquarium was undertaken with staff wearing a full face mask to prevent ingestion of water and animal faeces, while vacuuming faecal material from gravel. Gloves were worn when handling sick or potentially infected rays. Staff with open wounds, cuts or abrasions had these covered prior to diving in the exhibit. An ultraviolet steriliser was installed on the water pipe outlet in the exhibit pool to prevent the spread of bacteria from water exiting the ray exhibit into the connecting aquarium exhibit. Water was also treated with ultraviolet and chlorinated at 20 ppm, prior to water exiting into the external marine environment. The substrate in the ray exhibit was changed to larger sized gravel, facilitating easier vacuuming and improved removal of faecal and food waste material to minimise bacterial load in the system.

Antimicrobial therapy Antimicrobial therapy began after Streptococcus agalactiae was diagnosed as the cause of death of rays in May 2009. Rays presenting with similar clinical signs of disease were treated by intramuscular (IM) injection with either enrofloxacin (10 mg kg-1 sid IM), ceftazidime (20 mg kg-1 sid IM) or florfenicol (40 mg kg-1 every three days IM). Treatment with antibiotics did not alter the course of the disease and almost all of the rays showing clinical signs subsequently died, despite antimicrobial therapy. In an attempt to provide antibiotics as a preventative measure, all rays were subjected to an immersion bath with oxytetracycline continuously at a dose rate of 50 mg L-1 for ten days. Following immersion bathing, amoxicillin was administered to rays orally, at a dose of 25 mg kg-1 sid, administered in a gelatin capsule hidden inside fresh fish. However, compliance was poor and medication appeared unpalatable, as the rays either refused the medicated food, or regurgitated the capsule after ingesting the food.

Pathology findings Necropsies of rays with clinical signs of disease showed marked splenomegaly, Author Manuscript hepatomegaly, congestion of tissues ventrally with widespread petechial haemorrhages on the ventral integument, swelling of the dorsal cranium, and hyperaemia of the brain meninges (Fig. 1). Histopathology revealed that all examined rays were affected with a similar assortment of lesions indicative of acute bacteraemia, including granulocytic (infiltrate of

neutrophils and/or heterophils) to predominantly mononuclear infiltrate (including lymphocytes and macrophages), with myocarditis, meningoencephalitis, branchitis, and a variety of splenic lesions, all with intra-lesional Gram positive cocci, in pairs or chains (Figs 2, 3 and 4).

Myocarditis was the most common lesion, occurring in 20/21 (95%) rays for which microscopic evaluation could be conducted on the heart (Fig. 2). Myocarditis was consistently mild to moderate in severity, despite the severity of inflammation in other organs, and leukocyte infiltrate was most pronounced in perivascular areas. In 11/20 (55%) rays with myocarditis the infiltrate consisted of a mixture of granulocytes and mononuclear cells, and in the remaining rays with myocarditis the infiltrate was mononuclear. Gram positive cocci were visualised, either extracellularly between myofibres, or intracellularly in the cytoplasm of leucocytes (Fig. 3). In 6/20 (30%) of rays with myocarditis, epicarditis involving similar leukocyte infiltrate was also present.

Meningoencephalitis was the second most frequent lesion, occurring in 20/23 (87%) rays (Fig. 4). The severity was mild in 9/20 (45%), moderate in 9/20 (45%), and severe in 2/20 (10%). Leukocyte infiltrate expanded the leptomeninges and tended to extend into perivascular areas of the brain parenchyma with increasing severity. In 15/20 (75%) rays with meningoencephalitis the infiltrate was mononuclear whereas in 5/20 (25%) the infiltrate consisted of a mixture of granulocytes and mononuclear leukocytes. Meningoencephalitis typically had a generalized distribution, progressing from patchy to diffuse with increasing severity, and tending to be most pronounced lateral to the cerebellum and diencephalon. Meningoencephalitis was often accompanied by areas of gliosis and increased perivascular clear space suggestive of oedema. Severe meningoencephalitis also featured intravascular leucocytosis, thrombosis, and occasionally foci of parenchymal necrosis. Splenic lesions were relatively common, occurring in 10/22 (46%) rays, including severe congestion 3/10 (30%), mild to moderate mixed splenitis 3/10 (30%), and peri-ellipsoid histiocytosis 4/10 (40%).

Author Manuscript Branchitis affected 4/17 (24%) rays for which microscopic evaluation could be conducted on the gills. In these cases there was severe congestion of venous sinuses of gill filaments. A mixture of granulocytes and mononuclear leukocytes were within the venous sinuses and interstitium of the gill filaments and there were occasional foci of lamellar epithelial cell

hyperplasia. Other lesions observed in the study population included a mild mononuclear interstitial nephritis (n = 4), mild perivascular hepatitis (n = 2), moderate multifocal mixed hepatitis (n = 2), moderate mixed enteritis (n = 1), and severe mixed orchitis (n = 1).

Gram positive cocci, present in pairs or chains, were identified histologically in the tissues of all rays examined histologically (Figs. 2, 3 & 4). Bacteria could usually be identified in H&E stained sections, but when in low numbers, evaluation of Gram stained sections facilitated their identification. The number of bacteria present varied greatly among study rays and did not correlate well with overall lesion severity. Some rays had very mild lesions with large numbers of cocci, suggestive of peracute bacteremia. Where present, bacteria were usually within inflamed areas, often within the cytoplasm of leukocytes, or free within vascular spaces. Occasionally, bacteria were within organs that beared no or very mild lesions, in which case they were usually free within vascular spaces. Organs in which bacteria were identified included brain (n=19), heart (n=18), spleen (n=12), gill (n=5), liver (n=3), kidney (n = 2), intestine (n=1), and gonad (n=1).

Clinical pathology Cytology of fluid aspirated from dorsal cranial swellings of two rays, showed a highly proteinaceous background, with a minimal amount of blood, and mild heterophilic inflammation and, in one case, numerous intracellular and extracellular coccoid bacteria that were commonly present in long chains (Fig. 5). There were far fewer mononuclear inflammatory cells and moderate numbers of degenerate unidentifiable cells.

Bacteriology Bacteriology undertaken at the commercial veterinary pathology laboratory identified from each case, a catalase- negative, non-haemolytic Streptococcus sp., including Streptococcus pluranimalium. The ten isolates sent to a referral Queensland Government Veterinary Laboratory in Townsville, Queensland, confirmed the identification of every Streptococcus sp. isolate as Streptococcus agalactiae. A coccoid bacterium was isolated in pure culture from various organs and tissues including the brain, heart, kidney, liver, spleen, intestine and Author Manuscript oviduct. Bacterial growth was seen after 24 hours incubation at 25oC. The organism grew well at 25oC but was severely inhibited at 37oC. Colonies were non-haemolytic, opaque and mucoid in appearance and adhered strongly to blood agar. The organism occurred singly, in pairs, or in chains in serum broth culture, and was Gram positive. Antibiotic sensitivities

showed all isolates were sensitive to ampicillin, clindamycin, cotrimoxazole, enrofloxacin, erythromycin and tetracycline, all with intermediate or total resistance to gentamycin. In the 2010 epizootic, there were 3 cases where Photobacterium damselae was also isolated, however, intra-lesional Gram positive cocci were still visualised on histology in each case.

Bacterial molecular serotyping, PCR and sequencing Nucleic acid sequence analysis of the 16S-23S ribosomal DNA (rDNA) intergenic spacers of all S. agalactiae isolates obtained from rays showed all bacterial isolates had 100% homology with a non-haemolytic S. agalactiae. The whole genome BLASTn analysis of the cps locus with reference sequences revealed all ray isolates belong to serotype Ib. All ray strains had identical cps regions, with cpsA to cpsD, cpsF-cpsG, cpsL, and neuB to neuA being conserved amongst isolates (Delamare- Deboutteville 2014; Bowater 2015).

Ray cps loci were 15616 bp in size same as the cps of S. agalactiae isolated from deceased wild Qld grouper, javelin grunter and giant catfish species from north Queensland (data not shown). Comparative full genomic sequencing of the ray cps loci also showed they were smaller in size compared to human, canine, feline, or crocodilian cps sequences, and the ray cps were larger in size than the bovine cps (Delamare-Debouteville 2014; Bowater 2015).

Single nucleotide polymorphisms (SNPs) analysis of the cps sequences of the ray isolates revealed two identical SNPs found in the shovelnose ray (Aptychotrema rostrata) isolate, mangrove whipray (Himantura granulata) isolate, and the two estuary stingrays (Dasyatis fluviorum) isolate. These two SNPs are non-synonymous and result in an amino acid change, with an isoleucine being replaced by a valine in cpsC, and with a threonine being replaced by a proline in cpsE. This places the elasmobranch isolates in a single cluster distinct from the other Australian piscine isolates. Further molecular characterisation using MLST revealed all ray isolates belong to the piscine lineage sequence type ST-261 (Table III).

DISCUSSION Author Manuscript Streptococcus agalactiae appears to be an infectious disease of wild marine fish in Australia. S. agalactiae has already been reported causing deaths of wild adult Queensland grouper (Epinephelus lanceolatus), and other marine fish including javelin grunter (Pomadasys kaaken), giant sea catfish (Netuma thalassina), and squaretail mullet (Ellochelon vaigensis)

in north Queensland (Bowater et al. 2012). Here we have described two separate mortality epizootics caused by S. agalactiae affecting several species of rays from an exhibit in a commercial marine aquarium in south east Queensland.

The first disease epizootic occurred in 2009, affecting a mixture of ray species, in the exhibit. Although most of the rays that died from streptococcosis were wild and locally caught estuary rays, originating from the Moreton bay region in south east Queensland, some of the affected rays had originated from north Queensland, where S. agalactiae mortalities of wild Queensland grouper were concurrently occurring. Interestingly, a wild mangrove whipray (Himantura granulata) that was purchased from a licensed fish collector in north Queensland, was introduced into the population a few months prior to the first disease epizootic that occurred in 2009. This ray was placed into quarantine in late October 2008, and subsequently introduced into the exhibit in late January, along with other local-caught species of wild rays. It is possible this ray acted as a subclinical carrier of the bacterium and a point source for the epizootic, as it later succumbed to infection.

Molecular serotyping and MLST confirmed the S. agalactiae isolates obtained from sick and deceased rays from both disease epizootics, were unrelated to human and bovine isolates, as the serotype and ST of the marine isolates are different from local terrestrial isolates. They form a homogenous group that belongs to a piscine lineage, serotype Ib, and sequence type ST-261 (Delomare-Deboutteville 2014; Bowater 2015). This is reassuring from a public safety perspective as these ST and serotypes have not been found to cause disease in humans or terrestrial animals. The identical point mutations in the cps operon sequences found in all the ray isolates suggest passage amongst these animals and possible strain movement through translocation of animals from north Queensland. ST-261 has predominantly been found associated with disease epizootics occurring in farmed fish, in particular, with various tilapia and hybrid tilapia species. S. agalactiae ST-261 was first reported in 1988 from farmed Nile tilapia (Oreochromis niloticus) outbreaks in Israel (Eldar, Bejerano & Bercovier 1994), and has subsequently been isolated from diseased tilapia, from disease epizootics occurring in other countries, as Nile tilapia were progressively cultured elsewhere (Musa et al. 2009; Mian Author Manuscript et al. 2009; Lusiastuti et al. 2014; Lusiastuti 2009; Huang et al. 2013; Hernández et al. 2009; Anshary et al. 2014; Amal et al. 2013; Amal et al. 2012). Serotype Ib has also been previously reported from a wild ray (Dasyatis sp.) from a wild fish kill epizootic that occurred in the Gulf of Mexico in 1972, and from frogs (Plumb et al. 1974, Hetzel et al.

2003; Elliot et al. 1990). Serotype Ib (ST-261), was similarly isolated from sick, deceased wild giant Queensland grouper, and other marine fish, from wild epizootics occurring in northern Queensland in 2008, 2009 and 2010 (Bowater et al. 2012; Bowater 2015). Possible routes of infection and transmission for S. agalactiae infection in rays may include horizontal infection, through water-born exposure, or oral transmission through ingestion of contaminated feed, as documented for fish and other animals (Ren et al. 2014; Hetzel et al. 2003; Bowater 2015; Delamare-Deboutteville et al. 2015). In the present study, some rays had inflammation and S. agalactiae infection in the soft tissues of the dorsal cranium, suggestive of involvement of endolymphatic ducts, which bacteria could ascend to infect the brain and meninges. Rays may also become infected by inoculation, via trauma (potentially inoculated into wounds created by PIT tagging or spine clipping), or by direct contact during breeding or other interactions. Rays may have been sub-clinically infected when introduced into the exhibit, or may have naturally acquired S. agalactiae through ingestion of contaminated prey (fish, crustaceans) or water. Thawed frozen fish fed to rays, water and gravel substrate from the ray exhibit were consistently sampled, yet all were culture-negative for Streptococcus sp. during both epizootics (data not shown).

Host stress factors leading to immunosuppression and increased susceptibility to infection were likely influential in the occurrence of the present S. agalactiae epizootics. Many of the rays in the affected exhibit had been translocated from their natural habitat. Those originating from far north Queensland would have required acclimation to a new environment. Furthermore, the rays were handled for clipping of tail barbs and were initially kept at higher than normal stocking densities. In addition, a pump malfunction resulting in an episode of gas bubble disease, occurred in the exhibit two months prior to the first epizootic. High stocking density, handling, and changes in environmental or poor water quality parameters, including water temperature and dissolved oxygen levels, have all been observed as important stress factors involved in other epizootics of streptococcosis in fish (Eldar et al. 1995; Bromage, Thomas & Owens 1999; Evans, Shoemaker & Klesius 2003; Evans et al. 2006a; Hernández et al. 2009). Crowding of fish due to high stocking density may also directly facilitate horizontal transmission of pathogenic Streptococcus sp. (Evans et al. 2003). Author Manuscript

Water temperature may be an important factor for the occurrence of S. agalactiae mortality epidemics. Both of the present epizootics occurred during the cooler winter months, similar to wild epizootics of grouper mortalities in north Queensland. The first epizootic occurred in

late May continuing through to July 2009, and the second epizootic occurred in late July through to September 2010, both during the winter months. Similarly, the epizootics of streptococcosis in wild Queensland grouper occurred during the winter months, including periods when sea water temperatures were cooler than average (Bowater et al. 2012). This is in contrast to S. agalactiae outbreaks in wild fish of the Northern hemisphere which mostly occurred in spring or summer (Plumb et al. 1974, Evans et al. 2002). The contrast could be a reflection of differential impact of water temperature on the immune function of various host species, or environmental requirements of diverse S. agalactiae strains.

In general, S. agalactiae infection in fish typically results in lesions indicative of bacteraemia, that is, disseminated multifocal and multi-organ foci of inflammation, especially involving brain, eye, heart, kidney, liver, and spleen (Hernández et al. 2009, Evans et al. 2006a, Bowater et al. 2012; Baya et al. 1990). However, the nature of the inflammatory response varies among host species, ranging from granulomatous, to mononuclear, to granulocytic or necrotising (Edelsten & Pegram 1974; Zappulli et al. 2005; Musa et al. 2009; Liu et al. 2014; Ren et al. 2014). Rays in the present study showed a mixture of granulocytic to mononuclear inflammatory lesions, consistent with pathology shown by juvenile Queensland grouper inoculated with S. agalactiae (Bowater 2015; Delamare-Deboutteville et al. 2015). In contrast, pathology observed from wild, adult Queensland grouper, showed a predominately granulomatous inflammation of organs and tissues. (Bowater et al. 2012). It is unclear if the varying character of inflammatory infiltrates reflects differences in virulence factor expression of S. agalactiae, chronicity of infection, or species/age-specific distinctions in host immune response.

Streptococcosis should be a differential diagnosis in mortality epizootics of rays, both in the wild and in captivity, especially if accompanied by neurological signs. When S. agalactiae is suspected, diagnosticians should take care to include sampling brain, heart and spleen for histopathology as these tissues seemed to most consistently show lesions in the present epizootics. Gram stain and culture is recommended even where characteristic lesions are lacking, as numbers of bacteria observed histologically did not correlate with lesion severity. Author Manuscript A laboratory experienced in the culturing and identification of aquatic animal bacterial pathogens may be required if initial bacterial identification is equivocal. In our case series, a Gram-negative bacterium, Photobacterium damselae was also cultured from the internal organs of three rays, but co-infection was ruled out, since only Gram positive cocci were

histologically observed in lesions. P. damselae were likely post mortem commensals that overgrew S. agalactiae during bacterial culture. P. damselae is a bacterium that is present in marine water, and is a well-known cause of bacteraemia in fish and rays. It has previously been isolated from the blood of healthy rays (Toranzo, Magariños & Romalde 2005; Mylniczenko et al. 2007; Pedersen et al. 2009; Tao, Bullard & Arias 2014). Veterinarians examining sick marine rays or other fish exhibiting gross overt clinical signs of bacterial septicaemia, should therefore consider both pathogens as potential candidates on their list of differential diagnoses. Our observations emphasize the importance of histopathological diagnosis together with cytology and Gram staining to differentiate bacterial causes of disease.

Given the findings of the present study, marine aquaria maintaining captive collections of fish or elasmobranchs should be aware of potential risk for streptococcosis when introducing wild-caught animals, especially when fish are sourced from geographical areas where the disease has been previously diagnosed. Routine health testing and screening of animals prior to inter-state translocation in the wholesale fish industry and in the ornamental fish industry in Australia is largely lacking (Whittington & Chong 2007). The present study emphasises the risk that capture and translocation of wild fish of unknown health status may pose to aquarium businesses without stricter policy and regulation regarding translocation of wild marine animals within and between states. If streptococcosis is diagnosed in an aquarium setting, strict biosecurity procedures should be implemented to prevent spread to other marine exhibits or to wild populations, and to prevent exposure to humans.

A variety of antibiotics were unsuccessful in treating this disease in affected rays despite in- vitro sensitivity. These antibiotics also failed to prevent the spread of the disease throughout the population and did not seem to alter the course of the disease outbreaks. Vaccination was safe and effective as disease subsided soon after and has not reappeared since in the marine aquarium. To the authors’ knowledge this is the first time vaccination against a bacterial disease has been implemented in captive rays.

Author Manuscript The ecological impacts of this disease on marine ecosystems are unknown. The high mortlaities that incurred in captivity, and the poor host specificity known to exist for S. agalactiae, raise concern for the level of impact on ecosystem health. However, the degree of mortality observed in the present study is probably unlikely to occur naturally because of the

presence of several stress factors and artificial population density that are unlikely to be replicated in the wild.

In summary, this study describes a new setting for S. agalactiae mortality epizootics, an infectious disease of wild marine fish and now captive rays of Queensland. For more accurate epidemiological tracing and inference of evolutionary history, a larger genomic repertoire needs to be analysed, screening important virulence factors and performing SNP’s and phylogeny analysis on the complete genomes of those ray isolates. This will allow us to determine the source of infection, and the relatedness of the Australian S. agalactiae piscine isolates compared to overseas strains, enabling us to fully understand the true diversity and geographical distribution of the S. agalactiae piscine isolates in Australia.

ACKNOWLEDGEMENTS The authors would like to thank the divers, aquarists and veterinary nurses at Sea World who assisted with the treatments, post-mortems and vaccinations of the rays. Veterinary laboratory staff of the previous Department of Agriculture & Fisheries, Tropical & Aquatic Animal Health Laboratory, including; Dr Annette Thomas, Dr Judy Forbes-Faulkner, Andrew Fisk, Virna Duffy, Stacey Valdeter, Helen Smith, Chris Wright, Naomi Hooper, Liz Kulpa, Jenny Stanford and Kerri Dyer are thanked for their laboratory technical assistance. Dr Nouri Ben Zakour is thanked for genome sequencing work done on ray isolates that was done as part of a previous PhD and FRDC research project (Delamare-Deboutteville 2014; Bowater 2015). Author Manuscript

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Fig. 1. Gross pathology of a ray with bacterial septicaemia from infection with Streptococcus agalactiae. Note marked hepatomegaly (small arrow) protruding from dissected ventral abdominal surface, and widespread coalescing petechial and ecchymotic haemorrhages on the ventral surface (large arrow).

Author Manuscript Fig. 2. Heart; White spotted eagle ray (Aetobatus ocellatus). Leucocyte infiltrate (L) extends between myofibres (M). Most of the infiltrate comprises mononuclear

leucocytes, whereas granulocytes are in minority (arrows). 1000X magnification. (H&E).

Fig. 3. Heart; White spotted eagle ray (Aetobatus ocellatus). Gram positive cocci are extracellular (arrow) and within the cytoplasm of leucocytes (arrowheads). 1000X magnification. (Brown-Hopps Gram stain).

Fig. 4. Hindbrain; Estuary ray (Dasyatis fluviorum). Leucocyte infiltrate (L) severely expands the meninges of the hindbrain (B). 400X magnification. (H&E).

Fig. 5. Aspirate from a dorsal cranial swelling; Mangrove whip ray (Himantura granulata). Heterophil-predominant (arrow heads) inflammation with numerous coccoid bacteria, commonly seen in chains (arrows). Erythrocytes (*) and degenerate unidentifiable cells (#) were also present. 1000X magnification. Quick Dip. Author Manuscript

Table I. GenBank accession No. of the complete cps sequences of the reference strain for each existing serotype.

Author Manuscript

TABLE II Summary of ray mortalities and numbers infected with Streptococcus agalactiae from two mortality epizootics at a commercial

No. examined No. by No. examined bacteriology Species (N, %) Epidemic Date Mortalities by (no. culture (N#, %) histology positive SA, PD) ER EST MWR WSER BSMR

May- 11 8 2 1 0 0 1 July, 10 6 (6,0) (203, 5.4%) (74, 10.8%) (7, 28.6%) (5, 20.0%) (20, 0%) (48, 0%) 2009 July- 32 23 0 1 3 5 2 Oct, 14 11 (8,3) (170, 18.8%) (82, 28%) (2, 0%) (7, 14.3%) (13, 23.1%) (37, 13.5%) 2010

43 31 2 2 3 5 Total 24 17 (14,3) (252, 17.1%) (105, 29.5%) (7, 28.6%) (8, 25.0%) (22, 13.6%) (55, 9.1%)

marine aquarium in south east Queensland.

SA = Streptococcus agalactiae PD = Photobacterium damselae N#= total number of animals in the pool Author Manuscript # denotes some of the total numbers of animals were pups, separated to an adjacent pool N = total number of specific ray species in the exhibit

% = % confirmed dead from infection with Streptococcus agalactiae ER = Estuary ray (Dasyatis fluviorum) EST = Eastern shovelnose ray (Aptychotrema rostrata) MWR = Mangrove whipray (Himantura granulata) WSER = White spotted eagle ray (Aetobatus narinari) BSMR = Blue spotted mask ray (Neotrygon kuhlii) Author Manuscript

Table III. Allelic profile of the multilocus sequence type (ST-261) of the ray isolates Allelic profile serotype ST adhP pheS atr glnA sdhA glck tkt Ib 261 54 17 31 4 26 25 19 Abbreviations: alcohol dehydrogenase, adhP; phenylalanyl tRNA synthetase, pheS; glutamine transporter protein, atr; glutamine synthetase, glnA; serine dehydratase, sdhA; glucose kinase, glcK; and transketolase, tkt. Author Manuscript