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1 Western College of Veterinary Medicine, Saskatoon, Canada
2 Marine Biological Laboratory, Woods Hole, MA
* Corresponding author: rsmol{at}mbl.edu
Cuttlefish of the genus Sepia (S. officinalis, S. apama, and S. pharaonis) have been cultured at the Marine Resources Center (MRC) of the Marine Biological Laboratory since 1992. The objectives of this retrospective study were to identify common causes of morbidity and mortality in the cuttlefish populations maintained at the MRC, and to describe the histological appearance of those lesions. Such information can be used in developing more effective methods of diagnosis, prevention, and treatment.
Necropsy cases were selected for inclusion in this study if bacterial cultures had been obtained at necropsy. Bacterial cultures had been obtained from 53 necropsies performed between March 1999 and June 2003. Those culture samples were taken from the digestive gland, kidney sac, or gonad and were plated on marine brain heart infusion medium (1).
Retrospective examination of archived necropsy cases of Sepia spp. showed that mortality was commonly associated with bacterial cultures positive for Vibrio alginolyticus, a marine bacterium routinely found in coastal waters, sediment, and culture systems (2, 3). Of the 53 cases in which bacterial cultures were taken, 33 were positive for V. alginolyticus.
Of the 33 animals with cultures positive for the bacterium, archived histological sections were available in 19 cases. The sections were paraffin-embedded fixed tissues, sectioned at 6 µm and stained with hematoxylin and eosin (4). Examination of these sections showed that the most commonly affected tissues were the kidney, branchial heart appendage, branchial heart, and gill (42%). A high incidence of infection (47%) was noted in the reproductive organs (nidamental gland, accessory nidamental gland, and gonads). All animals with reproductive lesions were older than 9 months. Of the 19 included cases, 16 (84%) had some form of epidermal ulceration, with 7 classified as moderate to severe, and 9 as mild. No reaction was detected in the digestive gland of any animal. Examination of Gram-stained tissue sections (3) confirmed the presence of gram-negative bacteria in the infected foci.
Histopathological examination of sections showed that, based on the appearance of the response as identified in the tissues, the sepiod inflammatory reaction to V. alginolyticus occurred in one of three distinct forms. The first was multifocal, necrotizing, granuloma-like lesions (0.95–1.4 mm diameter), most often seen in reproductive tissues such as the testicular ducts and accessory nidamental glands (Fig. 1A, B). The second form consisted of multifocal, necrotizing, granulomatous-like inflammation that resulted in subtle lesions (0.1–0.25 mm diameter) containing small numbers of hemocytes and bacteria, and was predominately found in the gills of affected animals. Finally, multifocal, necrotizing, granulomatous-like inflammation of intermediate size was commonly found in branchial heart and its appendage (0.2–0.7 mm diameter) (Fig. 1C).
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Bacteria causing the observed lesions may have accessed the circulation via epidermal ulcerations, but ulceration was not recognized in all animals and was only mild in others, so the potential for other routes should not be ignored. In the circulation, the bacteria lodged in the branchial heart, resulting in foci of moderate inflammation and necrosis. Most other lesions appear to have been spread by bacterial seeding of organs that receive circulation from the branchial heart. Thus, bacteria-laden hemolymph that passed from the heart directly to the gill and branchial heart appendage resulted in foci of inflammation. The branchial heart appendage forms an ultrafiltrate that is discharged into the kidney sac and bathes the kidney fronds; the epithelium of the kidney fronds modifies the filtrate before it exits the body through a renal pore. Bacteria can proliferate in the filtrate and attack the suspended kidney fronds. Hemocytes, which mount the inflammatory response, migrate from vessels within the connective core of the kidney fronds to the site of infection.
These results show that V. alginolyticus is a pathogen of significance in sepoids cultured at the MRC. V. alginolyticus is common in coastal waters and is therefore likely to be present in the facility’s seawater supply (2). This pathogen has been associated with tank surfaces in culture systems (5). Comparisons between bacterial populations on wild-caught and laboratory-reared squid, Lolliguncula brevis, showed that animals reared in the laboratory had higher total numbers of bacteria. The increase was primarily due to Vibrio spp., including V. alginolyticus (6). In that study, the increase in bacteria was not linked to the ulceration of the epidermis. Similarly, V. alginolyticus has caused disease in juvenile red abalone, Haliotis rufescens, but large numbers can be found on the foot of otherwise healthy individuals (5). Such findings suggest that direct infection of the epidermis is an unlikely pathogenesis for V. alginolyticus in cuttlefish. Routes of infection probably include infection secondary to ulceration, especially if the injury is caused by jetting into tank walls colonized by the bacteria. V. alginolyticus has also been found on the carapace of copepods (7). Crustaceans, including amphipods, are the primary food fed to cuttlefish cultured at the MRC, and thus may be an important source of infection. Finally, over half of the studied cuttlefish with reproductive-associated lesions were at or near the upper limit of their life span (8), suggesting that senescent tissues are more susceptible to this pathogen and are a potential point of initiation for a systemic infection.
This retrospective study shows that systemic V. alginolyticus is a common pathogen in Sepia spp. cultured at the MRC. A number of potential routes of infection exist but, as in most aquatic animal diseases, stress and husbandry likely play an important role in the occurrence of disease. Better sepoid husbandry methods may help reduce or prevent this disease from occurring. Additionally, the data collected in this study indicate that, when systemic bacterial disease is suspected in Sepia spp., the branchial heart and branchial heart appendage should be examined histologically, and bacterial cultures should be obtained from the renal sac.
Literature Cited
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