Journal of Fish Diseases 2014
doi:10.1111/jfd.12254
Diseases of captive yellow seahorse Hippocampus kuda Bleeker, pot-bellied seahorse Hippocampus abdominalis Lesson and weedy seadragon Phyllopteryx taeniolatus ` de) (Lace´pe 3, M Kummrow4, V LePage1, J Young2, C J Dutton2, G Crawshaw2, J A Pare D J McLelland5, P Huber1, K Young1, S Russell1, L Al-Hussinee1 and J S Lumsden1 1 2 3 4 5
Fish Pathology Laboratory, Department of Pathobiology, University of Guelph, Guelph, ON, Canada Toronto Zoo, Scarborough, ON, Canada Wildlife Conservation Society, Bronx, NY, USA Zoo Hannover GmbH, Hannover, Germany Zoos South Australia, Adelaide, SA, Australia
Abstract
Seahorses, pipefish and seadragons are fish of the Family Syngnathidae. From 1998 to 2010, 172 syngnathid cases from the Toronto Zoo were submitted for post-mortem diagnostics and retrospectively examined. Among the submitted species were yellow seahorses Hippocampus kuda Bleeker (n = 133), potbellied seahorses Hippocampus abdominalis Lesson (n = 35) and weedy seadragons Phyllopteryx taeniolatus (Lace´pe`de; n = 4). The three most common causes of morbidity and mortality in this population were bacterial dermatitis, bilaterally symmetrical myopathy and mycobacteriosis, accounting for 24%, 17% and 15% of cases, respectively. Inflammatory processes were the most common diagnoses, present in 117 cases. Seven neoplasms were diagnosed, environmental aetiologies were identified in 46 cases, and two congenital defects were identified. Keywords: disease, seadragon, seahorse, syngnathid.
Introduction
Seahorses, pipefish and seadragons are fish of the Family Syngnathidae, which includes over 295 Correspondence J S Lumsden, Fish Pathology Laboratory, Department of Pathobiology, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada (e-mail: [email protected]) Ó 2014 John Wiley & Sons Ltd
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species (Froese & Pauly 2011). Syngnathids have attracted attention for decades due to their unique morphology, remarkable camouflage ability and the distinctive phenomenon whereby males give birth to live young. For many years, syngnathids have been collected from the wild for curiosities, exhibition in aquaria and traditional medicine. There are increasing data demonstrating that the current practice of wild syngnathid collection is unsustainable (Martin-Smith & Vincent 2006). Due to their charismatic nature and their vulnerability, syngnathids have become a flagship species for their respective ecosystems across the globe (MartinSmith & Vincent 2006; Shokri, Gladstone & Jelbart 2009). They have provided a focus for efforts towards sustainable use of marine resources, with international trade controls implemented in 2004, the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) Appendix II (Martin-Smith & Vincent 2006). A suggested alternative to wild-caught animals is captive culture or syngnathid aquaculture. While this has been successful for some species of syngnathids, it has not been shown to be cost-effective (Koldewey & Martin-Smith 2010). Captive breeding and rearing of many species of syngnathids remains a challenge due to disease from various pathogens, inappropriate nutrition and environmental conditions. There is a paucity of literature
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on syngnathid disease in comparison with other areas of syngnathid research. This report describes the causes of morbidity and mortality of three species of captive syngnathids held at the Toronto Zoo, Ontario, Canada, from 1998 until 2010: the yellow seahorse Hippocampus kuda, pot-bellied seahorse Hippocampus abdominalis and weedy seadragon Phyllopteryx taeniolatus (Lace´pe`de). A better understanding of the types of disease that affect this characteristic group of fish will improve future diagnostic procedures. This precedes the implementation of husbandry modifications, disease prevention, palliative care and treatment, with the ultimate benefit of improved survival and welfare in captivity. Materials and methods
From 1998 to 2010, 172 syngnathids from the Toronto Zoo were submitted for post-mortem diagnostics and were examined retrospectively. Among the submitted species were yellow seahorses Hippocampus kuda (n = 133), pot-bellied seahorses Hippocampus abdominalis (n = 35) and weedy seadragons P. taeniolatus (n = 4). These fish were held in a variety of marine enclosures (salinity 22–27 g L 1; Instant Ocean) at the Toronto Zoo in Scarborough, Ontario, Canada. All tanks had biofiltration systems (Hagen Aquaclear and Hagen Bio Foam; Hagen Inc.) without ozone or ultraviolet sterilization. Water temperatures ranged from 18 to 20 °C for the pot-bellied seahorses and 24–28 °C for the weedy seadragons and yellow seahorses (Thermal Compact Heaters; Hagen Inc.), and all fish were kept on a 13-h day/11-h night cycle. Water quality targets were as follows: no detectable ammonia or nitrite, and nitrate 70%) progressed from a severe ulcerative bacterial dermatitis. The organs most commonly affected were liver, kidney, gastrointestinal tract and in one case the epicardium. Lesions consisted mainly of necrosis centred on blood vessels of these organs with an intense collar of large numbers of macrophages filled with necrotic debris, fewer lymphocytes and intralesional bacteria. A small number of these cases did involve Gram-positive bacteria. Fifteen fish had a branchitis associated with bacteria; however, three of these had a mixed infection that also involved ciliated protozoans. The most common presentation involved extensive necrosis of lamellae and entire filaments, which were surrounded by extensive mats of filamentous but also morphologically mixed bacteria (Fig. 2). In affected gills, there were small to moderate numbers of macrophages filled with debris and other leucocytes within the central venous sinus and pillar cell channel and interstitial epithelium. Autolysis of the gills was common. There were two cases of epitheliocystis, both of which occurred in weedy seadragons. Multiple (>5 per gill arch) circular to ovoid intracytoplasmic
Figure 1 Female Hippocampus kuda with a hyperaemic tail or ‘red-tail’. Inset. Severe ulcerative skin lesion with adherent and invasive filamentous bacteria (H&E).
Journal of Fish Diseases 2014
bacterial microcolonies (approximately 25– 100 lm diameter), which were acid-fast negative but positive with a Pierce Van der Kamp stain, were present within the gill lamellar epithelium (Fig. 3). These microcolonies were also found within the epithelium lining the nasal pit, and the branchial and oral cavities. In the gastrointestinal tract lumen, the presence of large numbers of morphologically mixed bacteria was a common finding due to excessive bacterial growth on or within ingested food. In one case, a bacterial enteritis, presumably an extension of the bacterial overgrowth described above, was identified (1/172) (not shown). A population of short Gram-negative bacterial rods was noted that invaded and elevated the intestinal epithelium from the basement membrane, and this lesion was associated with epithelial cell swelling and necrosis. These bacteria were also seen within the lamina propria and underlying submucosa, and there was mild to moderate expansion of both layers by oedema. In cases of feed-associated bacterial overgrowth, large numbers of morphologically mixed bacteria were restricted to the intestinal lumen and the epithelium was either intact or autolysed. There were also typically copious food items in the intestinal lumen that contained similar bacterial populations (not shown). Parasitic. Seven fish were diagnosed with cryptosporidial enteritis. In these cases, the intestinal lumen was dilated and there were diffuse epithelial necrosis and attenuation with moderate numbers of both epicellular (~5 lm basophilic blebs) and
Figure 2 Necrotic branchial filaments and lamellae are surrounded by mats of filamentous bacteria mixed with bacteria of varied morphology (H&E). Ó 2014 John Wiley & Sons Ltd
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Figure 3 Numerous intracytoplasmic bacterial microcolonies within branchial lamellar epithelial cells that expand and distort lamellar structure (PVK stain).
intraepithelial (~1 lm eosinophilic and basophilic forms of apicomplexans consistent with Cryptosporidium sp. (Fig. 4). The connective tissue and blood vessels of the lamina propria and submucosa were expanded by oedema. Within all layers of the intestine and surrounding foci of necrosis, there were small numbers of morphologically mixed inflammatory cells and debris-filled macrophages. While the presence of rare protozoa was common in this population of syngnathids, protozoal dermatitis was determined to be a significant contributor to morbidity and/or mortality in 13 fish. Of these cases, nine protozoan infestations occurred in combination with a bacterial dermatitis. The organisms were teardrop-shaped ciliated protozoa, averaging 8 by 15 lm. Mild disease
Figure 4 Intracellular and epicellular forms of suspected cryptosporidial parasites associated with an attenuated and flattened intestinal epithelium with mild necrosis (H&E).
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Journal of Fish Diseases 2014
involved epithelial erosion, or mild necrosis, with the presence of ciliates along the affected epithelial surface. In severe infections, bacteria and occasionally fungi were also involved. In these cases, ulceration of the epithelium and superficial dermis and invasion of ciliates into the underlying musculature were widespread (not shown). Occasionally, in epizootics of mortality involving multiple newborn seahorses, protozoa were the only infectious agent present. The shape and size of these ciliated protozoa suggested a Uronema-like scuticociliate. Two cases involving mortality of multiple seahorse fry due to dermatitis with intralesional filamentous bacteria and Uronema-like protozoa also had a separate and distinctive protozoan present. These protozoa had eosinophilic circular bodies, 2–3 lm in diameter containing small basophilic nuclei. They were observed within dermal musculature and along the ulcerated epidermis (Fig. 5). Small numbers of macrophages in these lesions contained phagocytosed protozoans. Viral. A suspected viral enteritis was found to affect both adults and juvenile H. abdominalis. The disease was most apparent in younger fish, which experienced a higher mortality rate that occurred acutely with minimal clinical signs. The suspected viral enteritis was a problem for several months in 1 year and these lesions have not appeared since. The lesions considered typical of a viral infection were a markedly dilated intestinal lumen, a lack of luminal folds and an attenuated
Figure 5 Ulcerative dermatitis with numerous unidentified protozoa invading deeply within the dermis. Mats of filamentous bacteria and a Uronema-like parasite are also present within this lesion. Ó 2014 John Wiley & Sons Ltd
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epithelium with the absence of a visible aetiological agent on light microscopy. Mild necrosis of epithelial cells and submucosal oedema were also present (Fig. 6). The lesions were more severe in the younger animals. TEM revealed a few intracytoplasmic, non-enveloped viral-like particles up to 180 nm in size, which would be consistent with an iridovirus (Fig. 7). Neoplastic Seven neoplasms and two neoplastic-like lesions were identified in eight individuals consisting of one P. taeniolatus, one H. abdominalis and six H. kuda (LePage et al. 2012). The overall prevalence of neoplasia in syngnathids at the Toronto Zoo was 4.1%. Types of neoplasms included cardiac rhabdomyosarcoma, renal adenoma, renal adenocarcinoma, renal round cell tumour, exocrine pancreatic carcinoma and intestinal carcinoma. Environmental Gas bubble disease. Gas bubble disease (GBD) was characterized by the gross observation of excess gas accumulation in tissues. In syngnathids at the Toronto Zoo, gas was found to accumulate most commonly in subcutaneous tissues anywhere along the body and within the brood pouch. These fish presented with buoyancy problems and were often distressed and floating at the surface or had lost their ability to right themselves. Light
Figure 6 Intestinal cross-section with marked dilation of the intestinal lumen and thinning of the epithelium. Inset: Higher magnification of the thinned epithelium with mild necrosis and the absence of an aetiological agent (H&E).
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Journal of Fish Diseases 2014
experienced more than one event as muscle lesions of different degrees of chronicity could be found in the same animals. Regeneration, displayed as rowing of nuclei, was not prominent. Variation in myofibre diameter, likely compounded by atrophy due to starvation, was prominent. While infectious agents were not directly associated with these lesions, concurrent disease was very common. Twenty-two of 29 cases with bilaterally symmetrical myopathy also had dermatitis (bacterial and/or parasitic), but some fish also were affected with cryptosporidiosis, mycobacteriosis, non-acid-fast bacteraemia, GBD and neoplasia. Figure 7 Intestinal epithelial cell with brush border to the left and viral particles (arrow), approximately 180 nm in size.
microscopy was not always a useful aid in the diagnosis of GBD as artefactual lifting of the epidermis and separation along tissue planes commonly confounded interpretations. Over 85% of cases (13/15) with GBD also had a concurrent disease. The most common concurrent disease was a bacterial dermatitis, which based on the clinical history, occurred as a sequella to rupture of the gas bullae, which was precipitated by struggling to right. Bilaterally symmetrical myopathy. A myopathy was observed in H. kuda only (29/172 cases). Antemortem clinical signs were muscle weakness, as evidenced by decreased strength of prey striking or complete anorexia, inability to remain upright in the water column and lethargy. These fish were often found laterally recumbent at the bottom of the tank or with an abnormally bent neck and were euthanased due to their inability to eat. Tube feeding was attempted with some of these fish; however, the stress of frequent handling was counterproductive to the positive effects of this treatment. Under light microscopy, the myopathy was bilaterally symmetrical and was present in the axial musculature, mainly the epaxial muscles of the nuchal region. Three stages of the myopathy were noted: an early myopathy, with myofibre hypereosinophilia and loss of striation but without detectable hypercellularity (Fig. 8); an active ‘myopathy’ with large numbers of satellite cells and macrophages; and a chronic or resolving myopathy with myocyte sheath remnants and small condensed regenerating myofibres and with few macrophages. Some animals had likely Ó 2014 John Wiley & Sons Ltd
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Congenital Two congenital defects were observed in this population of syngnathids. The first was in a 2-yearold H. kuda with an expansive 200- to 250-lm blood-filled mass that effaced the myocardium at the apex of the ventricle and extended from the ventricular lumen to the epicardium (LePage et al. 2012). The second congenital defect was observed in an adult female H. abdominalis. On gross examination, there appeared to be duplication of the ovipositor (Fig. 9). Microscopically, there were two gravid ovaries, which were equal in size. Each ovarian duct appeared to exit through its own ovipositor rather than merging to a single one along midline. The ovipositors were located on each side, lateral to the expected location (Fig. 9). This finding did not appear to be associated with morbidity or mortality.
Figure 8 Skeletal muscle (cervical region of epaxial muscles) affected by a suspect nutritional myopathy with marked myofibre diameter variation and hypereosinophilia. Inset: Typical bilaterally symmetrical pattern of lesions (H&E).
Journal of Fish Diseases 2014
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Figure 9 Ovipositor duplication in Hippocampus abdominalis, showing left and right lateral views. Each space on the bottom ruler represents one millimetre.
Discussion
Inflammatory Bacterial. Most bacteria that have been isolated from syngnathids were from wild-caught clinically healthy animals or from captive animals with clinical signs of disease. Clinically significant bacterial diseases described from syngnathids were mostly limited to mycobacteriosis (Bombardini et al. 2006) and vibriosis (Alcaide et al. 2001; Tendencia 2004; Bombardini et al. 2006; Balcazar et al. 2010; Martins et al. 2010). However, Aeromonas spp, Tenacibaculum maritimum and Tsukamurella paurometabola have also been associated with morbidity and mortality (Bombardini et al. 2006). Non-acid-fast bacterial dermatitis was the most common cause of morbidity and mortality in this population of fish, accounting for almost 25% of cases. Lesions were often associated with filamentous bacteria, morphologically similar to Flavobacteriaceae, infiltrated within these erosive to ulcerative lesions. Due to the high prevalence in this population, further investigation of the aetiologies associated with this condition is warranted. As in other teleosts, mycobacteriosis in syngnathids is caused by atypical or non-tuberculous species, for example Mycobacterium chelonae, M. fortuitum and M. marinum (Koldewey 2005). Reported lesions in teleosts range from skin ulcers Ó 2014 John Wiley & Sons Ltd
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to systemic granulomatous disease and bacteraemia (Bombardini et al. 2006), similar to the range of lesions noted in the present study. The consequences of ulcerative skin lesions in fish are more significant than in mammals as the epidermis and dermis not only act as a barrier against pathogens but also limit significant shifts of water from the fish to the external environment in the case of marine fish. When skin ulcerations cover too large a surface area for the fish to compensate, they can quickly lead to mortality (Ferguson 2006). In the present study, there was one instance of bacterial enteritis that was interpreted to be clinically significant. Bacteria within these lesions were not characterized; however, gastroenteritis and necrotizing enteritis have been associated with Vibrio species in other teleosts (Lee, Liu & Chuang 2002), for example V. carchariae in summer flounder (Paralichthys dentatus Linnaeus) (Soffientino et al. 1999) and groupers Epinephelus coioides (Hamilton) (Yii, Yang & Lee 1997). In healthy captive H. kuda, the predominant bacterial flora was found to belong to the genus Vibrio (Tanu et al. 2011). Necrotizing bacterial branchitis in syngnathids was suspected to be associated with Flavobacteriaceae, due to the filamentous morphology of bacteria (Bombardini et al. 2006), and the lesions present in the seven cases noted in the present study were very similar. They were not unlike the
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branchial necrosis in freshwater teleosts caused by Flavobacterium columnare (Bernadet 1989; Decostere, Haesebrouck & Devriese 1998). There are two published reports of epitheliocystis in syngnathid gills: one in a weedy seadragon (Langdon, Elliott & MacKay 1991) that was later associated with a chlamydial agent (Meijer et al. 2006) and the second in a greater pipefish (Syngnathus acus Linnaeus) (Longshaw, Green & Feist 2004). Two cases of epitheliocystis with large numbers of very large inclusions were observed in weedy seadragons in the present study. The previously reported cases had few inclusions and were considered to be incidental and without clinical significance (Langdon et al. 1991; Longshaw et al. 2004). The numerous and very large inclusions noted in the present study in affected fish may have had some clinical impact. It is possible that seadragons have a propensity for the development of epitheliocystis or a susceptibility to chlamydia-like organisms, but firm conclusions cannot be drawn from such low case numbers. Parasitic. Parasites are the most extensively documented group of infectious agents in syngnathids. More specifically, metazoans noted to infect syngnathids include acanthocephalans (Braicovich, Gonzalez & Tanzola 2005), nematodes (Longshaw et al. 2004), monogeneans (Holliman 1963; Appleby 1996; Williams et al. 2008; Paladini et al. 2010; Vaughan et al. 2010), trematodes (Longshaw et al. 2004) and myxosporeans (Vincent & Clifton-Hadley 1989; Longshaw et al. 2004; Garner et al. 2008). Protozoans reported to infect syngnathids include ciliophorans such as scuticociliates (Cheung, Nigrelli & Ruggieri 1980; Umehara, Kosuga & Hirose 2003; Rossteuscher et al. 2008), peritrichs (Longshaw et al. 2004) and apicomplexans (Upton et al. 2000). The parasites observed in the present population of syngnathids were consistent with the literature. Overall, the clinical significance of parasitic diseases of syngnathids in this study was likely minimal with the exception of cryptosporidiosis and the Uronemalike ciliates affecting the newborn seahorses. Scuticociliates, primarily Uronema marinum, and to a lesser extent Philasterides dicentrarchi, commonly parasitize syngnathids and have been identified in seahorses (Cheung et al. 1980), seadragons (Umehara et al. 2003; Rossteuscher et al. 2008) and pipefish (Bombardini et al. 2006). These agents have been associated with erosive Ó 2014 John Wiley & Sons Ltd
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lesions of the skin and gill and often invade into muscle and systemically (Cheung et al. 1980; Umehara et al. 2003; Bombardini et al. 2006; Rossteuscher et al. 2008; Stidworthy 2008). This is in agreement with the infection of Uronema-like organisms found in the Toronto Zoo syngnathids, in which deep invasion by ciliates was observed on occasion; however, this was usually associated with widespread dermal ulceration and concurrent bacterial dermatitis. Cryptosporidiosis was a more significant pathogen in this population of syngnathids, causing regionally extensive lesions in the intestinal mucosa of animals with no other evidence of concurrent disease. Two apicomplexans have been documented in syngnathids: Eimeria syngnathi in Syngnathus abaster (Risso), formerly S. nigrolineatus (Yakimoff & Gousseff 1936), and Eimeria phillopterycis in P. taeniolatus (Upton et al. 2000). The clinical signs that have been noted were anorexia and stunted growth (Upton et al. 2000), and lesions included various stages of the parasite encysted within the gastrointestinal mucosa and submucosa with a mild inflammatory response (Upton et al. 2000). While tissue damage in these cases was not extensive, a heavy burden could cause malabsorption and poor growth. Cryptosporidia may have similar pathogenicity; however, they have been found to be largely incidental in some species of fish such as tilapia, but can be pathogenic in young fish (Lumsden 2006; Ryan 2010). Due to the young age and severity of lesions in the affected seahorses, the cryptosporidia likely contributed to the morbidity and mortality. Brine shrimp (Artemia) have been implicated as the mode of transmission of Cryptosporidium in cultured fish (Mendez-Hermida, Gomez-Couso & Ares-Mazas 2007), and this may have been the case for the small outbreak of cryptosporidiosis in this population of seahorses. Mycotic. In the present study, mycotic infections were restricted to the skin and were only found in syngnathids with other causes of disease. The stress of captivity, artificial systems and concurrent infections may contribute to immunosuppression and predispose captive syngnathids to opportunistic infections (Nyaoke et al. 2009). While fungi are often considered secondary invaders in fish (Roberts, Palmeiro & Weber 2009), there have been reports of mycoses causing mortalities in seahorses. Microsporidiosis caused
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by Glugea heraldi has been well described in wild-caught Hippocampus erectus (Perry) (Blasiola 1979; Vincent & Clifton-Hadley 1989; Bombardini et al. 2006) causing over 95% mortality within 6 months in wild-caught animals (Vincent & Clifton-Hadley 1989). Glugea heraldi causes multifocal cysts that eventually coalesce and rupture, predisposing to secondary bacterial infections. Two Exophiala species have been documented to cause a disseminated infection in both species of seadragons (Nyaoke et al. 2009). Lesions consisted of parenchymal and vascular necrosis with systemic fungal invasion. Of these cases, 65% had epithelial ulcerations with associated mats of fungal hyphae invading the dermis, hypodermis, fascia and skeletal muscle (Nyaoke et al. 2009). The vascular invasion and visceral lesions were associated with only Exophila sp.; however, it is unknown whether the inciting lesion was bacterial, fungal or traumatic (Nyaoke et al. 2009). In the present set of cases, systemic fungal infections were not noted and the hyphae present were always seen with other infectious agents, suggesting opportunistic invasion of abnormal epidermis. Viral. No virus has been identified from syngnathids to date. In this study, some juvenile animals were noted to have lesions consistent with but not pathognomonic for a viral intestinal infection. These lesions included blunting of intestinal folds, flattening of the remaining enterocytes, luminal distension, epithelial necrosis and the absence of other pathogens. Small numbers of intracytoplasmic viral-like particles were noted in the intestinal epithelium upon examination by TEM. Cell lines derived from syngnathids, of which there are presently none to our knowledge, could facilitate future research into viral causes of disease. Neoplastic Eight spontaneous neoplasms have been described in syngnathids including a brood pouch fibrosarcoma (Willens, Dunn & Frasca 2004), cardiac rhabdomyosarcoma, renal adenoma, renal adenocarcinoma, renal round cell tumour (suspect lymphoma), exocrine pancreatic carcinoma and intestinal carcinoma (LePage et al. 2012). While neoplasia is not a leading cause of mortality, the types of neoplasms affecting this species should be Ó 2014 John Wiley & Sons Ltd
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documented to monitor genetic and environmental predisposing factors. Environmental Gas bubble disease. Gas bubble disease (GBD) or gas supersaturation is common in fish (Speare 1990; Koppang & Bjerk as 2006), and syngnathids seem particularly susceptible. Mortality due to acute uncomplicated GBD is rare; however, rupture of emphysematous bullae beneath the epidermis can predispose to secondary bacterial infections (LePage, personal observation). Gas accumulation in documented cases of GBD in syngnathids occurred mainly as subcutaneous emphysema along the tail and in the brood pouch, less commonly in the head region or as an overinflated swim bladder (Bombardini et al. 2006; Lin, Lin & Huang 2010). In this study, one particular episode was linked to accidental rapid water heating and gas accumulation occurred in subcutaneous tissues along the tail and in the brood pouch. After correction of the supersaturation, the condition resolved spontaneously in most fish; however, active removal of gas from brood pouch was occasionally attempted. A second gas accumulation phenomenon occurred in seahorse fry that developed an overinflated swim bladder and struggled at the surface with excessive buoyancy; this was also often referred to as GBD (LePage, personal observation). Syngnathids are physoclists, and after fry rise to the surface to fill the swim bladder, the pneumatic duct closes within 24 h (Genten, Terwinghe & Danguy 2009). Some fry remain overly buoyant at the surface, perhaps due to overinflation; however, the pathogenesis is unknown. The use of Kreisel tanks or fine mesh at the surface of the water to prevent fry from remaining at the surface after they have taken their first gulp of air reduced the number of overly buoyant syngnathid fry and improved survival rates (LePage, personal observation). Suspect nutritional myopathy. The most common causes of myopathy in animals include nutritional, exertional, congenital and infectious (Koller & Exon 1986; Wallace, Bush & Montali 1987; Hartup et al. 1999; Bassett & Currie 2003; Turnbull 2006). The most thoroughly investigated cause of myopathy is nutritional muscular dystrophy, linked to vitamin E and selenium deficiency (Koller & Exon 1986); however in barramundi,
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skeletal muscle myopathy was found to develop in potassium-deficient water (Partridge & Creeper 2004). Nutritional myopathy presents as muscle weakness, inability to eat and general failure to thrive (Koller & Exon 1986; Wallace et al. 1987; Hartup et al. 1999; Bassett & Currie 2003; Turnbull 2006; Van Vleet & Valentine 2007). Light microscopic lesions of nutritional myopathy are bilaterally symmetrical, with minimal inflammation. Myofibres are initially hypercontracted, as evidenced by the loss of striation, and are large, rounded and have hypereosinophilic and granular sarcoplasm. Later, macrophages can phagocytose these hypercontracted fibre segments along with other fragmented myofibres that may or may not have undergone mineralization (Van Vleet & Valentine 2007). The lesions were similar in H. kuda except that there was no or only rare mineralization, consistent with myopathies described in other teleosts (Holloway & Smith 1982; Turnbull 2006). In this study, H. kuda was the most susceptible as they were the only syngnathids to develop a myopathy despite similar diets and environmental conditions. This could also indicate that a diet suitable for a temperate water species such as H. abdominalis may not be adequate for a warmer water species like H. kuda. More research is required to determine the nutritional requirements of each syngnathid species in order to tailor diets accordingly. Congenital Most teleosts produce a large number of offspring, many of which have a high degree of exposure to the external environment during incubation. Both of these factors contribute to an increased frequency of congenital defects (Laale & Lerner 1981). Additionally, any defects are more likely to be observed, as the odds of survival are increased in captivity. Ovipositor duplication in the present report and the blood-filled cyst within the ventricular myocardium reported previously (LePage et al. 2012) are two of the first documented congenital defects reported in this family of fish. One other report describes a weedy seadragon with an absent or malformed swim bladder as observed by CT scan (Garland et al. 2002). While it is quite common to see alterations in gonads, such as ovotestes, in teleosts (Vethaak et al. 2002), Ó 2014 John Wiley & Sons Ltd
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external openings or structures are rarely duplicated or altered. Acknowledgements VL was funded in part by an Ontario Veterinary College Fellowship, a Natural Sciences and Engineering Research Council Discovery Grant (JSL), a CL Davis Fellowship and a John L. Pitts Recent Veterinary Graduate Scholarship from the World Aquatic Veterinary Medical Association. The work was funded in part by NSERC Discovery, OVC Pet Trust and the Toronto Zoo Endangered Species Fund. Thanks to Cindy Lee of the Toronto Zoo for her support of the project. References Alcaide E., Gil-Sanz C., Sanjuan E., Esteve E., Amaro C. & Silveira L. (2001) Vibrio harveyi causes disease in seahorse, Hippocampus sp. Journal of Fish Diseases 24, 311–313. Appleby C. (1996) Gyrodactylus syngnathi n. sp. (Monogenea: Gyrodactylidae) from the pipefish Syngnathus rostellatus Nilsson, 1855 (Syngnathiformes: Syngnathidae) from the Oslo Fjord. Norway. Systemic Parasitology 33, 131–134. Balcazar J.L., Gallo-Bueno A., Planas M. & Pintado J. (2010) Isolation of Vibrio alginolyticus and Vibrio splendidus from captive-bred seahorses with disease symptoms. Antonie van Leeuwenhoek 97, 207–210. Bassett D.I. & Currie P.D. (2003) The zebrafish as a model for muscular dystrophy and congenital myopathy. Human Molecular Genetics 12, R265–R270. Bernadet J.F. (1989) ‘Flexibacter columnaris’: first description in France and comparison with bacterial strains from other origins. Diseases of Aquatic Organisms 6, 37–44. Blasiola G.C. (1979) Glugea heraldi n. sp. (Microsporida, Glugeidae) from the seahorse Hippocampus erectus Perry. Journal of Fish Diseases 2, 493–500. Bombardini C., Florio D., Fichtel L. & Fioravanti M.L. (2006) The main disease of Syngnathidae in captivity. Ittiopatologia 3, 205–211. Braicovich P.E., Gonzalez R.A. & Tanzola R.D. (2005) First record of Corynosoma austral (Acanthocephala, Polymorphidae) parasitizing seahorse, Hippocampus sp. (Pisces, Syngnathidae) in Patagonia (Argentina). Acta Parasitologica 50, 145–149. Cheung P.J., Nigrelli R.F. & Ruggieri G.D. (1980) Studies on the morphology of Uronema marinum Dujardin (Ciliatea: Uronematidae) with a description of the histopathology of the infection in marine fishes. Journal of Fish Diseases 3, 295–303. Decostere A., Haesebrouck F. & Devriese L.A. (1998) Characterization of four Flavobacterium columnare (Flexibacter columaris) strains isolated from tropical fish. Veterinary Microbiology 62, 35–45.
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Ferguson H.W. (2006) Skin. In: Systemic Pathology of Fish (ed. by H.W. Ferguson), pp. 65–89. Iowa State University Press, Iowa, USA. Froese F. & Pauly D. (2011) FishBase. www.fishbase.org. Accessed: 8 June 2011. Garland M.R., Lawler L.P., Whitaker B.R., Walker I.D.F. & Corl F.M. (2002) Modern CT application in veterinary medicine. RadioGraphics 22, 55–62. Garner M.M., Atkinson S.D., Hallett S.L., Bartholomew J.L., Nordhausen R.W., Reed H., Adams L. & Whitaker B. (2008) Renal myxozoanosis in weedy sea dragons, Phyllopteryx taeniolatus (Lacepede), caused by Sinuolinea phyllopteryxa n. sp. Journal of Fish Diseases 31, 27–35. Genten F., Terwinghe E. & Danguy A. (2009) Swim and Gas Bladders. In: Atlas of Fish Histology (ed. by F. Genten, E. Terwinghe & A. Danguy), pp. 99–103. Science Publishers, New Hampshire, USA. Hartup B.K., Kollias G.V., Jacobsen M.C., Valentine B.A. & Kimber K.R. (1999) Exertional myopathy in translocated river otters from New York. Journal of Wildlife Diseases 35, 542–547. Holliman R.B. (1963) Gyrodactylus shorti, a new species of monogenetic trematode from the brood pouch of the southern pipefish, Syngnathus scovelli (Evermann and Kendall). Tulane Studies in Zoology and Botany 12, 83–86. Holloway H.L. & Smith C.E. (1982) A myopathy in North Dakota walleye, Stizostedion vitreum (Mitchell). Journal of Fish Diseases 5, 527–530. Koldewey H. (2005) Syngnathid health management. In: Seahorse Husbandry in Public Aquaria Manual (ed. by C.D. Bull & J.S. Mitchell), pp. 16–20, 32–33, 55. John D. Shedd Aquarium, Illinois, USA. Koldewey H.J. & Martin-Smith K.M. (2010) A global review of seahorse aquaculture. Aquaculture 302, 131–152. Koller L.D. & Exon J.H. (1986) The two faces of selenium – deficiency and toxicity – are similar in animals and man. Canadian Journal of Veterinary Research 50, 297–306. Koppang O.E. & Bjerk as E. (2006) The eye. In: Systemic Pathology of Fish (ed. by H.W. Ferguson), pp. 250–251. Iowa State University Press, Iowa, USA. Laale H.W. & Lerner W. (1981) Teratology and early fish development. American Zoologist 21, 517–533. Langdon J.S., Elliott K. & MacKay B. (1991) Epitheliocystis in the leafy sea-dragon. Australian Veterinary Journal 68, 244. Lee K.K., Liu P.C. & Chuang W.H. (2002) Pathogenesis of gastroenteritis caused by Vibrio carchariae in cultured marine fish. Marine Biotechnology 4, 267–277.
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Longshaw M., Green M.J. & Feist S.W. (2004) Histopathology of parasitic infections in greater pipefish, Syngnathus acus L., from an estuary in the UK. Journal of Fish Diseases 27, 245–248. Lumsden J.S. (2006) Gastrointestinal tract, swimbladder, pancreas and peritoneum. In: Systemic Pathology of Fish (ed. by H.W. Ferguson), pp. 169–175. Iowa State University Press, Iowa, USA. Martins M.L., Mouri~ no J.L.P., Fezer G.F., Buglione Neto C.C., Garcia P., Silva B.C., Jatoba A.S. & Vieira F.N. (2010) Isolation and experimental infection with Vibrio alginolyticus in the sea horse, Hippocampus reidi Ginsburg, 1933 (Osteichthyes: Syngnathidae) in Brazil. Brazilian Journal of Biology 70, 205–209. Martin-Smith K. & Vincent A.C.J. (2006) Exploitation and trade of Australian seahorses, pipehorses, sea dragons and pipefishes (Family Syngnathidae). Oryx 40, 141–151. Meijer A., Roholl P.J.M., Ossewaarde J.M., Jones B. & Nowak B.F. (2006) Molecular evidence for association of Chlamydiales bacteria with epitheliocystis in leafy seadragon (Phycodurus eques), silver perch (Bidyanus bidyanus), and barramundi (Lates calcarifer). Applied and Environmental Microbiology 72, 284–290. Mendez-Hermida F., Gomez-Couso H. & Ares-Mazas E. (2007) Possible involvement of Artemia as live diet in the transmission of cryptosporidiosis in cultured fish. Parasitology Research 101, 823–827. Nyaoke A., Weber E.S., Innis C., Stremme D., Dowd C., Hinckley L., Gorton T., Wickes B., Sutton D., de Hoog S. & Frasca S. Jr (2009) Disseminated phaeohyphomycosis in weedy seadragons (Phyllopteryx taeniolatus) and leafy seadragons (Phycodurus eques) caused by species of Exophiala, including a novel species. Journal of Veterinary Diagnostic Investigation 21, 69–79. Paladini G., Cable H., Fioravanti M.L., Faria P.J. & Shinn A.P. (2010) The description of Gyrodactylus corleonis sp. n. and G. neretum sp. n. (Platyhelminthes: Monogenea) with comments on other gyrodactylids parasitizing pipefish (Pisces: Syngnathidae). Folia Parasitologica 57, 17–30. Partridge G.J. & Creeper J. (2004) Skeletal myopathy in juvenile barramundi, Lates calcarifer (Bloch), cultured in potassium-deficient saline groundwater. Journal of Fish Diseases 27, 523–530. Roberts H.E., Palmeiro B. & Weber E.S. (2009) Bacterial and parasitic diseases of pet fish. Veterinary Clinics of North America – Exotic Animal Practice 12, 609–638. Rossteuscher S., Wenker C., Jermann T., Wahli T., Oldenberg E. & Schmidt-Posthaus H. (2008) Severe scuticociliate (Philasterides dicentrarchi) infection in a population of sea dragons (Phycodurus eques and Phyllopteryx taeniolatus). Veterinary Pathology 45, 546–550.
LePage V., Dutton C.J., Kummrow M., McLelland D.J., Young K. & Lumsden J.S. (2012) Neoplasia of captive yellow sea horses (Hippocampus kuda) and weedy sea dragons (Phyllopteryx taeniolatus). Journal of Zoo and Wildlife Medicine 43, 50–58.
Ryan U. (2010) Cryptosporidium in birds, fish and amphibians. Experimental Parasitology 124, 113–120.
Lin Q., Lin J. & Huang L. (2010) Effects of light intensity, stocking density and temperature on the air-bubble disease, survivorship and growth of early juvenile seahorse Hippocampus erectus Perry, 1810. Aquaculture Research 42, 91–98.
Shokri M.R., Gladstone W. & Jelbart J. (2009) The effectiveness of seahorses and pipefish (Pisces: Syngnathidae) as a flagship group to evaluate the conservation value of estuarine seagrass beds. Aquatic Conservation: Marine and Freshwater Ecosystems 19, 588–595.
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Soffientino B., Gwaltney T., Nelson D.R., Specker J.L., Mauel M. & Gomez-Chiarri M. (1999) Infectious necrotizing enteritis and mortality caused by Vibrio carchariae in summer flounder Paralichthys dentatus during intensive culture. Diseases of Aquatic Organisms 38, 201–210. Speare D.J. (1990) Histopathology and ultrastructure of ocular lesions associated with gas bubble disease in salmonids. Journal of Comparative Pathology 103, 421–432. Stidworthy M. (2008) Letter to the editor: scuticociliatosis in sea dragons. Veterinary Pathology 45, 957. Tanu D.D.D., Khandeparker R., Sreepada R., Sanaye S.V. & Pawar H.B. (2011) A study on bacteria associated with the intestinal tract of farmed yellow seahorse, Hippocampus kuda (Bleeker, 1852): characterization and extracellular enzymes. Aquaculture Research 43, 1–9. Tendencia E.A. (2004) The first report of Vibrio harveyi infection in the sea horse Hippocampus kuda Bleeker 1852 in the Philippines. Aquaculture Research 35, 1292–1294. Turnbull J. (2006) Musculoskeletal system. In: Systemic Pathology of Fish (ed. by H.W. Ferguson), pp. 289–307. Iowa State University Press, Iowa, USA. Umehara A., Kosuga Y. & Hirose H. (2003) Scuticociliata infection in the weedy sea dragon Phyllopteryx taeniolatus. Parasitology International 52, 165–168. Upton S.J., Stamper M.A., Osborn A.L., Mumford S.L., Zwick L., Kinsel M.J. & Overstreet R.M. (2000) A new species of Eimeria (Apicomplexa, Eimeriidae) from the weedy sea dragon Phyllpteryx taeniolatus (Osteichthyes: Syngnathidae). Diseases of Aquatic Organisms 43, 55–59. Van Vleet J.F. & Valentine B.A. (2007) Muscle and tendon. In: Pathology of Domestic Animals, vol. 1 (ed. by G.M. Maxie), pp. 236–242. Saunders Press, Ontario, Canada. Vaughan D.B., Christison K.W., Hansen H. & Shinn A.P. (2010) Gyrodactylus eyipayipi sp. n. (Monogenea:
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Gyrodactylidae) from Syngnathus acus (Syngnathidae) from South Africa. Folia Parasitologica 57, 11–15. Vethaak A.D., Lahr J., Kuiper R.V., Grinwis G.C.M., Rankouhi T.R., Giesy J.P. & Gerritsen A. (2002) Estrogenic effects in fish in the Netherlands: some preliminary results. Toxicology 181–182, 147–150. Vincent A.C.J. & Clifton-Hadley R.S. (1989) Parasitic infection of the seahorse (Hippocampus erectus) – a case report. Journal of Wildlife Diseases 25, 404–406. Wallace R.S., Bush M. & Montali R.J. (1987) Deaths from exertional myopathy at the National Zoological Park from 1975–1985. Journal of Wildlife Diseases 23, 454–462. Willens S., Dunn J.L. & Frasca S. Jr (2004) Fibrosarcoma of the brood pouch in an aquarium-reared lined seahorse (Hippocampus erectus). Journal of Zoo and Wildlife Medicine 35, 107–109. Williams S.R., Kritsky D.C., Dunnigan B., Lash R. & Klein P. (2008) Gyrodactylus pisculentus sp. n. (Monogenoidea: Gyrodactylidae) associated with mortality of the northern pipefish, Syngnathus fuscus (Syngnathiformes: Syngnathidae) at Woods Hole Science Aquarium. Folia Parasitologica 55, 265–269. Yakimoff W.L. & Gousseff W.F. (1936) Eimeria syngnathi n. sp. A new coccidium from the great pipefish (Syngnathus nigrolineatus). Journal of the Royal Microscopical Society 56, 376. Yii K.C., Yang T.I. & Lee K.K. (1997) Isolation and characterization of Vibrio carchariae, a causative agent of gastroenteritis in the groupers, Epinephelus coioides. Current Microbiology 35, 109–115. Received: 16 February 2014 Revision received: 23 March 2014 Accepted: 24 March 2014
doi:10.1111/jfd.12254
Diseases of captive yellow seahorse Hippocampus kuda Bleeker, pot-bellied seahorse Hippocampus abdominalis Lesson and weedy seadragon Phyllopteryx taeniolatus ` de) (Lace´pe 3, M Kummrow4, V LePage1, J Young2, C J Dutton2, G Crawshaw2, J A Pare D J McLelland5, P Huber1, K Young1, S Russell1, L Al-Hussinee1 and J S Lumsden1 1 2 3 4 5
Fish Pathology Laboratory, Department of Pathobiology, University of Guelph, Guelph, ON, Canada Toronto Zoo, Scarborough, ON, Canada Wildlife Conservation Society, Bronx, NY, USA Zoo Hannover GmbH, Hannover, Germany Zoos South Australia, Adelaide, SA, Australia
Abstract
Seahorses, pipefish and seadragons are fish of the Family Syngnathidae. From 1998 to 2010, 172 syngnathid cases from the Toronto Zoo were submitted for post-mortem diagnostics and retrospectively examined. Among the submitted species were yellow seahorses Hippocampus kuda Bleeker (n = 133), potbellied seahorses Hippocampus abdominalis Lesson (n = 35) and weedy seadragons Phyllopteryx taeniolatus (Lace´pe`de; n = 4). The three most common causes of morbidity and mortality in this population were bacterial dermatitis, bilaterally symmetrical myopathy and mycobacteriosis, accounting for 24%, 17% and 15% of cases, respectively. Inflammatory processes were the most common diagnoses, present in 117 cases. Seven neoplasms were diagnosed, environmental aetiologies were identified in 46 cases, and two congenital defects were identified. Keywords: disease, seadragon, seahorse, syngnathid.
Introduction
Seahorses, pipefish and seadragons are fish of the Family Syngnathidae, which includes over 295 Correspondence J S Lumsden, Fish Pathology Laboratory, Department of Pathobiology, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada (e-mail: [email protected]) Ó 2014 John Wiley & Sons Ltd
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species (Froese & Pauly 2011). Syngnathids have attracted attention for decades due to their unique morphology, remarkable camouflage ability and the distinctive phenomenon whereby males give birth to live young. For many years, syngnathids have been collected from the wild for curiosities, exhibition in aquaria and traditional medicine. There are increasing data demonstrating that the current practice of wild syngnathid collection is unsustainable (Martin-Smith & Vincent 2006). Due to their charismatic nature and their vulnerability, syngnathids have become a flagship species for their respective ecosystems across the globe (MartinSmith & Vincent 2006; Shokri, Gladstone & Jelbart 2009). They have provided a focus for efforts towards sustainable use of marine resources, with international trade controls implemented in 2004, the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) Appendix II (Martin-Smith & Vincent 2006). A suggested alternative to wild-caught animals is captive culture or syngnathid aquaculture. While this has been successful for some species of syngnathids, it has not been shown to be cost-effective (Koldewey & Martin-Smith 2010). Captive breeding and rearing of many species of syngnathids remains a challenge due to disease from various pathogens, inappropriate nutrition and environmental conditions. There is a paucity of literature
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on syngnathid disease in comparison with other areas of syngnathid research. This report describes the causes of morbidity and mortality of three species of captive syngnathids held at the Toronto Zoo, Ontario, Canada, from 1998 until 2010: the yellow seahorse Hippocampus kuda, pot-bellied seahorse Hippocampus abdominalis and weedy seadragon Phyllopteryx taeniolatus (Lace´pe`de). A better understanding of the types of disease that affect this characteristic group of fish will improve future diagnostic procedures. This precedes the implementation of husbandry modifications, disease prevention, palliative care and treatment, with the ultimate benefit of improved survival and welfare in captivity. Materials and methods
From 1998 to 2010, 172 syngnathids from the Toronto Zoo were submitted for post-mortem diagnostics and were examined retrospectively. Among the submitted species were yellow seahorses Hippocampus kuda (n = 133), pot-bellied seahorses Hippocampus abdominalis (n = 35) and weedy seadragons P. taeniolatus (n = 4). These fish were held in a variety of marine enclosures (salinity 22–27 g L 1; Instant Ocean) at the Toronto Zoo in Scarborough, Ontario, Canada. All tanks had biofiltration systems (Hagen Aquaclear and Hagen Bio Foam; Hagen Inc.) without ozone or ultraviolet sterilization. Water temperatures ranged from 18 to 20 °C for the pot-bellied seahorses and 24–28 °C for the weedy seadragons and yellow seahorses (Thermal Compact Heaters; Hagen Inc.), and all fish were kept on a 13-h day/11-h night cycle. Water quality targets were as follows: no detectable ammonia or nitrite, and nitrate 70%) progressed from a severe ulcerative bacterial dermatitis. The organs most commonly affected were liver, kidney, gastrointestinal tract and in one case the epicardium. Lesions consisted mainly of necrosis centred on blood vessels of these organs with an intense collar of large numbers of macrophages filled with necrotic debris, fewer lymphocytes and intralesional bacteria. A small number of these cases did involve Gram-positive bacteria. Fifteen fish had a branchitis associated with bacteria; however, three of these had a mixed infection that also involved ciliated protozoans. The most common presentation involved extensive necrosis of lamellae and entire filaments, which were surrounded by extensive mats of filamentous but also morphologically mixed bacteria (Fig. 2). In affected gills, there were small to moderate numbers of macrophages filled with debris and other leucocytes within the central venous sinus and pillar cell channel and interstitial epithelium. Autolysis of the gills was common. There were two cases of epitheliocystis, both of which occurred in weedy seadragons. Multiple (>5 per gill arch) circular to ovoid intracytoplasmic
Figure 1 Female Hippocampus kuda with a hyperaemic tail or ‘red-tail’. Inset. Severe ulcerative skin lesion with adherent and invasive filamentous bacteria (H&E).
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bacterial microcolonies (approximately 25– 100 lm diameter), which were acid-fast negative but positive with a Pierce Van der Kamp stain, were present within the gill lamellar epithelium (Fig. 3). These microcolonies were also found within the epithelium lining the nasal pit, and the branchial and oral cavities. In the gastrointestinal tract lumen, the presence of large numbers of morphologically mixed bacteria was a common finding due to excessive bacterial growth on or within ingested food. In one case, a bacterial enteritis, presumably an extension of the bacterial overgrowth described above, was identified (1/172) (not shown). A population of short Gram-negative bacterial rods was noted that invaded and elevated the intestinal epithelium from the basement membrane, and this lesion was associated with epithelial cell swelling and necrosis. These bacteria were also seen within the lamina propria and underlying submucosa, and there was mild to moderate expansion of both layers by oedema. In cases of feed-associated bacterial overgrowth, large numbers of morphologically mixed bacteria were restricted to the intestinal lumen and the epithelium was either intact or autolysed. There were also typically copious food items in the intestinal lumen that contained similar bacterial populations (not shown). Parasitic. Seven fish were diagnosed with cryptosporidial enteritis. In these cases, the intestinal lumen was dilated and there were diffuse epithelial necrosis and attenuation with moderate numbers of both epicellular (~5 lm basophilic blebs) and
Figure 2 Necrotic branchial filaments and lamellae are surrounded by mats of filamentous bacteria mixed with bacteria of varied morphology (H&E). Ó 2014 John Wiley & Sons Ltd
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Figure 3 Numerous intracytoplasmic bacterial microcolonies within branchial lamellar epithelial cells that expand and distort lamellar structure (PVK stain).
intraepithelial (~1 lm eosinophilic and basophilic forms of apicomplexans consistent with Cryptosporidium sp. (Fig. 4). The connective tissue and blood vessels of the lamina propria and submucosa were expanded by oedema. Within all layers of the intestine and surrounding foci of necrosis, there were small numbers of morphologically mixed inflammatory cells and debris-filled macrophages. While the presence of rare protozoa was common in this population of syngnathids, protozoal dermatitis was determined to be a significant contributor to morbidity and/or mortality in 13 fish. Of these cases, nine protozoan infestations occurred in combination with a bacterial dermatitis. The organisms were teardrop-shaped ciliated protozoa, averaging 8 by 15 lm. Mild disease
Figure 4 Intracellular and epicellular forms of suspected cryptosporidial parasites associated with an attenuated and flattened intestinal epithelium with mild necrosis (H&E).
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involved epithelial erosion, or mild necrosis, with the presence of ciliates along the affected epithelial surface. In severe infections, bacteria and occasionally fungi were also involved. In these cases, ulceration of the epithelium and superficial dermis and invasion of ciliates into the underlying musculature were widespread (not shown). Occasionally, in epizootics of mortality involving multiple newborn seahorses, protozoa were the only infectious agent present. The shape and size of these ciliated protozoa suggested a Uronema-like scuticociliate. Two cases involving mortality of multiple seahorse fry due to dermatitis with intralesional filamentous bacteria and Uronema-like protozoa also had a separate and distinctive protozoan present. These protozoa had eosinophilic circular bodies, 2–3 lm in diameter containing small basophilic nuclei. They were observed within dermal musculature and along the ulcerated epidermis (Fig. 5). Small numbers of macrophages in these lesions contained phagocytosed protozoans. Viral. A suspected viral enteritis was found to affect both adults and juvenile H. abdominalis. The disease was most apparent in younger fish, which experienced a higher mortality rate that occurred acutely with minimal clinical signs. The suspected viral enteritis was a problem for several months in 1 year and these lesions have not appeared since. The lesions considered typical of a viral infection were a markedly dilated intestinal lumen, a lack of luminal folds and an attenuated
Figure 5 Ulcerative dermatitis with numerous unidentified protozoa invading deeply within the dermis. Mats of filamentous bacteria and a Uronema-like parasite are also present within this lesion. Ó 2014 John Wiley & Sons Ltd
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epithelium with the absence of a visible aetiological agent on light microscopy. Mild necrosis of epithelial cells and submucosal oedema were also present (Fig. 6). The lesions were more severe in the younger animals. TEM revealed a few intracytoplasmic, non-enveloped viral-like particles up to 180 nm in size, which would be consistent with an iridovirus (Fig. 7). Neoplastic Seven neoplasms and two neoplastic-like lesions were identified in eight individuals consisting of one P. taeniolatus, one H. abdominalis and six H. kuda (LePage et al. 2012). The overall prevalence of neoplasia in syngnathids at the Toronto Zoo was 4.1%. Types of neoplasms included cardiac rhabdomyosarcoma, renal adenoma, renal adenocarcinoma, renal round cell tumour, exocrine pancreatic carcinoma and intestinal carcinoma. Environmental Gas bubble disease. Gas bubble disease (GBD) was characterized by the gross observation of excess gas accumulation in tissues. In syngnathids at the Toronto Zoo, gas was found to accumulate most commonly in subcutaneous tissues anywhere along the body and within the brood pouch. These fish presented with buoyancy problems and were often distressed and floating at the surface or had lost their ability to right themselves. Light
Figure 6 Intestinal cross-section with marked dilation of the intestinal lumen and thinning of the epithelium. Inset: Higher magnification of the thinned epithelium with mild necrosis and the absence of an aetiological agent (H&E).
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experienced more than one event as muscle lesions of different degrees of chronicity could be found in the same animals. Regeneration, displayed as rowing of nuclei, was not prominent. Variation in myofibre diameter, likely compounded by atrophy due to starvation, was prominent. While infectious agents were not directly associated with these lesions, concurrent disease was very common. Twenty-two of 29 cases with bilaterally symmetrical myopathy also had dermatitis (bacterial and/or parasitic), but some fish also were affected with cryptosporidiosis, mycobacteriosis, non-acid-fast bacteraemia, GBD and neoplasia. Figure 7 Intestinal epithelial cell with brush border to the left and viral particles (arrow), approximately 180 nm in size.
microscopy was not always a useful aid in the diagnosis of GBD as artefactual lifting of the epidermis and separation along tissue planes commonly confounded interpretations. Over 85% of cases (13/15) with GBD also had a concurrent disease. The most common concurrent disease was a bacterial dermatitis, which based on the clinical history, occurred as a sequella to rupture of the gas bullae, which was precipitated by struggling to right. Bilaterally symmetrical myopathy. A myopathy was observed in H. kuda only (29/172 cases). Antemortem clinical signs were muscle weakness, as evidenced by decreased strength of prey striking or complete anorexia, inability to remain upright in the water column and lethargy. These fish were often found laterally recumbent at the bottom of the tank or with an abnormally bent neck and were euthanased due to their inability to eat. Tube feeding was attempted with some of these fish; however, the stress of frequent handling was counterproductive to the positive effects of this treatment. Under light microscopy, the myopathy was bilaterally symmetrical and was present in the axial musculature, mainly the epaxial muscles of the nuchal region. Three stages of the myopathy were noted: an early myopathy, with myofibre hypereosinophilia and loss of striation but without detectable hypercellularity (Fig. 8); an active ‘myopathy’ with large numbers of satellite cells and macrophages; and a chronic or resolving myopathy with myocyte sheath remnants and small condensed regenerating myofibres and with few macrophages. Some animals had likely Ó 2014 John Wiley & Sons Ltd
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Congenital Two congenital defects were observed in this population of syngnathids. The first was in a 2-yearold H. kuda with an expansive 200- to 250-lm blood-filled mass that effaced the myocardium at the apex of the ventricle and extended from the ventricular lumen to the epicardium (LePage et al. 2012). The second congenital defect was observed in an adult female H. abdominalis. On gross examination, there appeared to be duplication of the ovipositor (Fig. 9). Microscopically, there were two gravid ovaries, which were equal in size. Each ovarian duct appeared to exit through its own ovipositor rather than merging to a single one along midline. The ovipositors were located on each side, lateral to the expected location (Fig. 9). This finding did not appear to be associated with morbidity or mortality.
Figure 8 Skeletal muscle (cervical region of epaxial muscles) affected by a suspect nutritional myopathy with marked myofibre diameter variation and hypereosinophilia. Inset: Typical bilaterally symmetrical pattern of lesions (H&E).
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Figure 9 Ovipositor duplication in Hippocampus abdominalis, showing left and right lateral views. Each space on the bottom ruler represents one millimetre.
Discussion
Inflammatory Bacterial. Most bacteria that have been isolated from syngnathids were from wild-caught clinically healthy animals or from captive animals with clinical signs of disease. Clinically significant bacterial diseases described from syngnathids were mostly limited to mycobacteriosis (Bombardini et al. 2006) and vibriosis (Alcaide et al. 2001; Tendencia 2004; Bombardini et al. 2006; Balcazar et al. 2010; Martins et al. 2010). However, Aeromonas spp, Tenacibaculum maritimum and Tsukamurella paurometabola have also been associated with morbidity and mortality (Bombardini et al. 2006). Non-acid-fast bacterial dermatitis was the most common cause of morbidity and mortality in this population of fish, accounting for almost 25% of cases. Lesions were often associated with filamentous bacteria, morphologically similar to Flavobacteriaceae, infiltrated within these erosive to ulcerative lesions. Due to the high prevalence in this population, further investigation of the aetiologies associated with this condition is warranted. As in other teleosts, mycobacteriosis in syngnathids is caused by atypical or non-tuberculous species, for example Mycobacterium chelonae, M. fortuitum and M. marinum (Koldewey 2005). Reported lesions in teleosts range from skin ulcers Ó 2014 John Wiley & Sons Ltd
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to systemic granulomatous disease and bacteraemia (Bombardini et al. 2006), similar to the range of lesions noted in the present study. The consequences of ulcerative skin lesions in fish are more significant than in mammals as the epidermis and dermis not only act as a barrier against pathogens but also limit significant shifts of water from the fish to the external environment in the case of marine fish. When skin ulcerations cover too large a surface area for the fish to compensate, they can quickly lead to mortality (Ferguson 2006). In the present study, there was one instance of bacterial enteritis that was interpreted to be clinically significant. Bacteria within these lesions were not characterized; however, gastroenteritis and necrotizing enteritis have been associated with Vibrio species in other teleosts (Lee, Liu & Chuang 2002), for example V. carchariae in summer flounder (Paralichthys dentatus Linnaeus) (Soffientino et al. 1999) and groupers Epinephelus coioides (Hamilton) (Yii, Yang & Lee 1997). In healthy captive H. kuda, the predominant bacterial flora was found to belong to the genus Vibrio (Tanu et al. 2011). Necrotizing bacterial branchitis in syngnathids was suspected to be associated with Flavobacteriaceae, due to the filamentous morphology of bacteria (Bombardini et al. 2006), and the lesions present in the seven cases noted in the present study were very similar. They were not unlike the
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branchial necrosis in freshwater teleosts caused by Flavobacterium columnare (Bernadet 1989; Decostere, Haesebrouck & Devriese 1998). There are two published reports of epitheliocystis in syngnathid gills: one in a weedy seadragon (Langdon, Elliott & MacKay 1991) that was later associated with a chlamydial agent (Meijer et al. 2006) and the second in a greater pipefish (Syngnathus acus Linnaeus) (Longshaw, Green & Feist 2004). Two cases of epitheliocystis with large numbers of very large inclusions were observed in weedy seadragons in the present study. The previously reported cases had few inclusions and were considered to be incidental and without clinical significance (Langdon et al. 1991; Longshaw et al. 2004). The numerous and very large inclusions noted in the present study in affected fish may have had some clinical impact. It is possible that seadragons have a propensity for the development of epitheliocystis or a susceptibility to chlamydia-like organisms, but firm conclusions cannot be drawn from such low case numbers. Parasitic. Parasites are the most extensively documented group of infectious agents in syngnathids. More specifically, metazoans noted to infect syngnathids include acanthocephalans (Braicovich, Gonzalez & Tanzola 2005), nematodes (Longshaw et al. 2004), monogeneans (Holliman 1963; Appleby 1996; Williams et al. 2008; Paladini et al. 2010; Vaughan et al. 2010), trematodes (Longshaw et al. 2004) and myxosporeans (Vincent & Clifton-Hadley 1989; Longshaw et al. 2004; Garner et al. 2008). Protozoans reported to infect syngnathids include ciliophorans such as scuticociliates (Cheung, Nigrelli & Ruggieri 1980; Umehara, Kosuga & Hirose 2003; Rossteuscher et al. 2008), peritrichs (Longshaw et al. 2004) and apicomplexans (Upton et al. 2000). The parasites observed in the present population of syngnathids were consistent with the literature. Overall, the clinical significance of parasitic diseases of syngnathids in this study was likely minimal with the exception of cryptosporidiosis and the Uronemalike ciliates affecting the newborn seahorses. Scuticociliates, primarily Uronema marinum, and to a lesser extent Philasterides dicentrarchi, commonly parasitize syngnathids and have been identified in seahorses (Cheung et al. 1980), seadragons (Umehara et al. 2003; Rossteuscher et al. 2008) and pipefish (Bombardini et al. 2006). These agents have been associated with erosive Ó 2014 John Wiley & Sons Ltd
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lesions of the skin and gill and often invade into muscle and systemically (Cheung et al. 1980; Umehara et al. 2003; Bombardini et al. 2006; Rossteuscher et al. 2008; Stidworthy 2008). This is in agreement with the infection of Uronema-like organisms found in the Toronto Zoo syngnathids, in which deep invasion by ciliates was observed on occasion; however, this was usually associated with widespread dermal ulceration and concurrent bacterial dermatitis. Cryptosporidiosis was a more significant pathogen in this population of syngnathids, causing regionally extensive lesions in the intestinal mucosa of animals with no other evidence of concurrent disease. Two apicomplexans have been documented in syngnathids: Eimeria syngnathi in Syngnathus abaster (Risso), formerly S. nigrolineatus (Yakimoff & Gousseff 1936), and Eimeria phillopterycis in P. taeniolatus (Upton et al. 2000). The clinical signs that have been noted were anorexia and stunted growth (Upton et al. 2000), and lesions included various stages of the parasite encysted within the gastrointestinal mucosa and submucosa with a mild inflammatory response (Upton et al. 2000). While tissue damage in these cases was not extensive, a heavy burden could cause malabsorption and poor growth. Cryptosporidia may have similar pathogenicity; however, they have been found to be largely incidental in some species of fish such as tilapia, but can be pathogenic in young fish (Lumsden 2006; Ryan 2010). Due to the young age and severity of lesions in the affected seahorses, the cryptosporidia likely contributed to the morbidity and mortality. Brine shrimp (Artemia) have been implicated as the mode of transmission of Cryptosporidium in cultured fish (Mendez-Hermida, Gomez-Couso & Ares-Mazas 2007), and this may have been the case for the small outbreak of cryptosporidiosis in this population of seahorses. Mycotic. In the present study, mycotic infections were restricted to the skin and were only found in syngnathids with other causes of disease. The stress of captivity, artificial systems and concurrent infections may contribute to immunosuppression and predispose captive syngnathids to opportunistic infections (Nyaoke et al. 2009). While fungi are often considered secondary invaders in fish (Roberts, Palmeiro & Weber 2009), there have been reports of mycoses causing mortalities in seahorses. Microsporidiosis caused
Journal of Fish Diseases 2014
by Glugea heraldi has been well described in wild-caught Hippocampus erectus (Perry) (Blasiola 1979; Vincent & Clifton-Hadley 1989; Bombardini et al. 2006) causing over 95% mortality within 6 months in wild-caught animals (Vincent & Clifton-Hadley 1989). Glugea heraldi causes multifocal cysts that eventually coalesce and rupture, predisposing to secondary bacterial infections. Two Exophiala species have been documented to cause a disseminated infection in both species of seadragons (Nyaoke et al. 2009). Lesions consisted of parenchymal and vascular necrosis with systemic fungal invasion. Of these cases, 65% had epithelial ulcerations with associated mats of fungal hyphae invading the dermis, hypodermis, fascia and skeletal muscle (Nyaoke et al. 2009). The vascular invasion and visceral lesions were associated with only Exophila sp.; however, it is unknown whether the inciting lesion was bacterial, fungal or traumatic (Nyaoke et al. 2009). In the present set of cases, systemic fungal infections were not noted and the hyphae present were always seen with other infectious agents, suggesting opportunistic invasion of abnormal epidermis. Viral. No virus has been identified from syngnathids to date. In this study, some juvenile animals were noted to have lesions consistent with but not pathognomonic for a viral intestinal infection. These lesions included blunting of intestinal folds, flattening of the remaining enterocytes, luminal distension, epithelial necrosis and the absence of other pathogens. Small numbers of intracytoplasmic viral-like particles were noted in the intestinal epithelium upon examination by TEM. Cell lines derived from syngnathids, of which there are presently none to our knowledge, could facilitate future research into viral causes of disease. Neoplastic Eight spontaneous neoplasms have been described in syngnathids including a brood pouch fibrosarcoma (Willens, Dunn & Frasca 2004), cardiac rhabdomyosarcoma, renal adenoma, renal adenocarcinoma, renal round cell tumour (suspect lymphoma), exocrine pancreatic carcinoma and intestinal carcinoma (LePage et al. 2012). While neoplasia is not a leading cause of mortality, the types of neoplasms affecting this species should be Ó 2014 John Wiley & Sons Ltd
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documented to monitor genetic and environmental predisposing factors. Environmental Gas bubble disease. Gas bubble disease (GBD) or gas supersaturation is common in fish (Speare 1990; Koppang & Bjerk as 2006), and syngnathids seem particularly susceptible. Mortality due to acute uncomplicated GBD is rare; however, rupture of emphysematous bullae beneath the epidermis can predispose to secondary bacterial infections (LePage, personal observation). Gas accumulation in documented cases of GBD in syngnathids occurred mainly as subcutaneous emphysema along the tail and in the brood pouch, less commonly in the head region or as an overinflated swim bladder (Bombardini et al. 2006; Lin, Lin & Huang 2010). In this study, one particular episode was linked to accidental rapid water heating and gas accumulation occurred in subcutaneous tissues along the tail and in the brood pouch. After correction of the supersaturation, the condition resolved spontaneously in most fish; however, active removal of gas from brood pouch was occasionally attempted. A second gas accumulation phenomenon occurred in seahorse fry that developed an overinflated swim bladder and struggled at the surface with excessive buoyancy; this was also often referred to as GBD (LePage, personal observation). Syngnathids are physoclists, and after fry rise to the surface to fill the swim bladder, the pneumatic duct closes within 24 h (Genten, Terwinghe & Danguy 2009). Some fry remain overly buoyant at the surface, perhaps due to overinflation; however, the pathogenesis is unknown. The use of Kreisel tanks or fine mesh at the surface of the water to prevent fry from remaining at the surface after they have taken their first gulp of air reduced the number of overly buoyant syngnathid fry and improved survival rates (LePage, personal observation). Suspect nutritional myopathy. The most common causes of myopathy in animals include nutritional, exertional, congenital and infectious (Koller & Exon 1986; Wallace, Bush & Montali 1987; Hartup et al. 1999; Bassett & Currie 2003; Turnbull 2006). The most thoroughly investigated cause of myopathy is nutritional muscular dystrophy, linked to vitamin E and selenium deficiency (Koller & Exon 1986); however in barramundi,
Journal of Fish Diseases 2014
skeletal muscle myopathy was found to develop in potassium-deficient water (Partridge & Creeper 2004). Nutritional myopathy presents as muscle weakness, inability to eat and general failure to thrive (Koller & Exon 1986; Wallace et al. 1987; Hartup et al. 1999; Bassett & Currie 2003; Turnbull 2006; Van Vleet & Valentine 2007). Light microscopic lesions of nutritional myopathy are bilaterally symmetrical, with minimal inflammation. Myofibres are initially hypercontracted, as evidenced by the loss of striation, and are large, rounded and have hypereosinophilic and granular sarcoplasm. Later, macrophages can phagocytose these hypercontracted fibre segments along with other fragmented myofibres that may or may not have undergone mineralization (Van Vleet & Valentine 2007). The lesions were similar in H. kuda except that there was no or only rare mineralization, consistent with myopathies described in other teleosts (Holloway & Smith 1982; Turnbull 2006). In this study, H. kuda was the most susceptible as they were the only syngnathids to develop a myopathy despite similar diets and environmental conditions. This could also indicate that a diet suitable for a temperate water species such as H. abdominalis may not be adequate for a warmer water species like H. kuda. More research is required to determine the nutritional requirements of each syngnathid species in order to tailor diets accordingly. Congenital Most teleosts produce a large number of offspring, many of which have a high degree of exposure to the external environment during incubation. Both of these factors contribute to an increased frequency of congenital defects (Laale & Lerner 1981). Additionally, any defects are more likely to be observed, as the odds of survival are increased in captivity. Ovipositor duplication in the present report and the blood-filled cyst within the ventricular myocardium reported previously (LePage et al. 2012) are two of the first documented congenital defects reported in this family of fish. One other report describes a weedy seadragon with an absent or malformed swim bladder as observed by CT scan (Garland et al. 2002). While it is quite common to see alterations in gonads, such as ovotestes, in teleosts (Vethaak et al. 2002), Ó 2014 John Wiley & Sons Ltd
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external openings or structures are rarely duplicated or altered. Acknowledgements VL was funded in part by an Ontario Veterinary College Fellowship, a Natural Sciences and Engineering Research Council Discovery Grant (JSL), a CL Davis Fellowship and a John L. Pitts Recent Veterinary Graduate Scholarship from the World Aquatic Veterinary Medical Association. The work was funded in part by NSERC Discovery, OVC Pet Trust and the Toronto Zoo Endangered Species Fund. Thanks to Cindy Lee of the Toronto Zoo for her support of the project. References Alcaide E., Gil-Sanz C., Sanjuan E., Esteve E., Amaro C. & Silveira L. (2001) Vibrio harveyi causes disease in seahorse, Hippocampus sp. Journal of Fish Diseases 24, 311–313. Appleby C. (1996) Gyrodactylus syngnathi n. sp. (Monogenea: Gyrodactylidae) from the pipefish Syngnathus rostellatus Nilsson, 1855 (Syngnathiformes: Syngnathidae) from the Oslo Fjord. Norway. Systemic Parasitology 33, 131–134. Balcazar J.L., Gallo-Bueno A., Planas M. & Pintado J. (2010) Isolation of Vibrio alginolyticus and Vibrio splendidus from captive-bred seahorses with disease symptoms. Antonie van Leeuwenhoek 97, 207–210. Bassett D.I. & Currie P.D. (2003) The zebrafish as a model for muscular dystrophy and congenital myopathy. Human Molecular Genetics 12, R265–R270. Bernadet J.F. (1989) ‘Flexibacter columnaris’: first description in France and comparison with bacterial strains from other origins. Diseases of Aquatic Organisms 6, 37–44. Blasiola G.C. (1979) Glugea heraldi n. sp. (Microsporida, Glugeidae) from the seahorse Hippocampus erectus Perry. Journal of Fish Diseases 2, 493–500. Bombardini C., Florio D., Fichtel L. & Fioravanti M.L. (2006) The main disease of Syngnathidae in captivity. Ittiopatologia 3, 205–211. Braicovich P.E., Gonzalez R.A. & Tanzola R.D. (2005) First record of Corynosoma austral (Acanthocephala, Polymorphidae) parasitizing seahorse, Hippocampus sp. (Pisces, Syngnathidae) in Patagonia (Argentina). Acta Parasitologica 50, 145–149. Cheung P.J., Nigrelli R.F. & Ruggieri G.D. (1980) Studies on the morphology of Uronema marinum Dujardin (Ciliatea: Uronematidae) with a description of the histopathology of the infection in marine fishes. Journal of Fish Diseases 3, 295–303. Decostere A., Haesebrouck F. & Devriese L.A. (1998) Characterization of four Flavobacterium columnare (Flexibacter columaris) strains isolated from tropical fish. Veterinary Microbiology 62, 35–45.
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Ferguson H.W. (2006) Skin. In: Systemic Pathology of Fish (ed. by H.W. Ferguson), pp. 65–89. Iowa State University Press, Iowa, USA. Froese F. & Pauly D. (2011) FishBase. www.fishbase.org. Accessed: 8 June 2011. Garland M.R., Lawler L.P., Whitaker B.R., Walker I.D.F. & Corl F.M. (2002) Modern CT application in veterinary medicine. RadioGraphics 22, 55–62. Garner M.M., Atkinson S.D., Hallett S.L., Bartholomew J.L., Nordhausen R.W., Reed H., Adams L. & Whitaker B. (2008) Renal myxozoanosis in weedy sea dragons, Phyllopteryx taeniolatus (Lacepede), caused by Sinuolinea phyllopteryxa n. sp. Journal of Fish Diseases 31, 27–35. Genten F., Terwinghe E. & Danguy A. (2009) Swim and Gas Bladders. In: Atlas of Fish Histology (ed. by F. Genten, E. Terwinghe & A. Danguy), pp. 99–103. Science Publishers, New Hampshire, USA. Hartup B.K., Kollias G.V., Jacobsen M.C., Valentine B.A. & Kimber K.R. (1999) Exertional myopathy in translocated river otters from New York. Journal of Wildlife Diseases 35, 542–547. Holliman R.B. (1963) Gyrodactylus shorti, a new species of monogenetic trematode from the brood pouch of the southern pipefish, Syngnathus scovelli (Evermann and Kendall). Tulane Studies in Zoology and Botany 12, 83–86. Holloway H.L. & Smith C.E. (1982) A myopathy in North Dakota walleye, Stizostedion vitreum (Mitchell). Journal of Fish Diseases 5, 527–530. Koldewey H. (2005) Syngnathid health management. In: Seahorse Husbandry in Public Aquaria Manual (ed. by C.D. Bull & J.S. Mitchell), pp. 16–20, 32–33, 55. John D. Shedd Aquarium, Illinois, USA. Koldewey H.J. & Martin-Smith K.M. (2010) A global review of seahorse aquaculture. Aquaculture 302, 131–152. Koller L.D. & Exon J.H. (1986) The two faces of selenium – deficiency and toxicity – are similar in animals and man. Canadian Journal of Veterinary Research 50, 297–306. Koppang O.E. & Bjerk as E. (2006) The eye. In: Systemic Pathology of Fish (ed. by H.W. Ferguson), pp. 250–251. Iowa State University Press, Iowa, USA. Laale H.W. & Lerner W. (1981) Teratology and early fish development. American Zoologist 21, 517–533. Langdon J.S., Elliott K. & MacKay B. (1991) Epitheliocystis in the leafy sea-dragon. Australian Veterinary Journal 68, 244. Lee K.K., Liu P.C. & Chuang W.H. (2002) Pathogenesis of gastroenteritis caused by Vibrio carchariae in cultured marine fish. Marine Biotechnology 4, 267–277.
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Soffientino B., Gwaltney T., Nelson D.R., Specker J.L., Mauel M. & Gomez-Chiarri M. (1999) Infectious necrotizing enteritis and mortality caused by Vibrio carchariae in summer flounder Paralichthys dentatus during intensive culture. Diseases of Aquatic Organisms 38, 201–210. Speare D.J. (1990) Histopathology and ultrastructure of ocular lesions associated with gas bubble disease in salmonids. Journal of Comparative Pathology 103, 421–432. Stidworthy M. (2008) Letter to the editor: scuticociliatosis in sea dragons. Veterinary Pathology 45, 957. Tanu D.D.D., Khandeparker R., Sreepada R., Sanaye S.V. & Pawar H.B. (2011) A study on bacteria associated with the intestinal tract of farmed yellow seahorse, Hippocampus kuda (Bleeker, 1852): characterization and extracellular enzymes. Aquaculture Research 43, 1–9. Tendencia E.A. (2004) The first report of Vibrio harveyi infection in the sea horse Hippocampus kuda Bleeker 1852 in the Philippines. Aquaculture Research 35, 1292–1294. Turnbull J. (2006) Musculoskeletal system. In: Systemic Pathology of Fish (ed. by H.W. Ferguson), pp. 289–307. Iowa State University Press, Iowa, USA. Umehara A., Kosuga Y. & Hirose H. (2003) Scuticociliata infection in the weedy sea dragon Phyllopteryx taeniolatus. Parasitology International 52, 165–168. Upton S.J., Stamper M.A., Osborn A.L., Mumford S.L., Zwick L., Kinsel M.J. & Overstreet R.M. (2000) A new species of Eimeria (Apicomplexa, Eimeriidae) from the weedy sea dragon Phyllpteryx taeniolatus (Osteichthyes: Syngnathidae). Diseases of Aquatic Organisms 43, 55–59. Van Vleet J.F. & Valentine B.A. (2007) Muscle and tendon. In: Pathology of Domestic Animals, vol. 1 (ed. by G.M. Maxie), pp. 236–242. Saunders Press, Ontario, Canada. Vaughan D.B., Christison K.W., Hansen H. & Shinn A.P. (2010) Gyrodactylus eyipayipi sp. n. (Monogenea:
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Gyrodactylidae) from Syngnathus acus (Syngnathidae) from South Africa. Folia Parasitologica 57, 11–15. Vethaak A.D., Lahr J., Kuiper R.V., Grinwis G.C.M., Rankouhi T.R., Giesy J.P. & Gerritsen A. (2002) Estrogenic effects in fish in the Netherlands: some preliminary results. Toxicology 181–182, 147–150. Vincent A.C.J. & Clifton-Hadley R.S. (1989) Parasitic infection of the seahorse (Hippocampus erectus) – a case report. Journal of Wildlife Diseases 25, 404–406. Wallace R.S., Bush M. & Montali R.J. (1987) Deaths from exertional myopathy at the National Zoological Park from 1975–1985. Journal of Wildlife Diseases 23, 454–462. Willens S., Dunn J.L. & Frasca S. Jr (2004) Fibrosarcoma of the brood pouch in an aquarium-reared lined seahorse (Hippocampus erectus). Journal of Zoo and Wildlife Medicine 35, 107–109. Williams S.R., Kritsky D.C., Dunnigan B., Lash R. & Klein P. (2008) Gyrodactylus pisculentus sp. n. (Monogenoidea: Gyrodactylidae) associated with mortality of the northern pipefish, Syngnathus fuscus (Syngnathiformes: Syngnathidae) at Woods Hole Science Aquarium. Folia Parasitologica 55, 265–269. Yakimoff W.L. & Gousseff W.F. (1936) Eimeria syngnathi n. sp. A new coccidium from the great pipefish (Syngnathus nigrolineatus). Journal of the Royal Microscopical Society 56, 376. Yii K.C., Yang T.I. & Lee K.K. (1997) Isolation and characterization of Vibrio carchariae, a causative agent of gastroenteritis in the groupers, Epinephelus coioides. Current Microbiology 35, 109–115. Received: 16 February 2014 Revision received: 23 March 2014 Accepted: 24 March 2014
