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Myocarditis in the common cuttlefish (Sepia oficinalis)
j. Comp. Path. 1990 Vol. 102
Myocarditis in the C o m m o n Cuttlefish
(Sepia oj~cinalis ) R. Reimschuessel*, M. K. Stoskopf] and R. O. Bennett+ *Department of Pathology, University of Maryland School of Medicine, 10 S. Pine Street, Baltimore, AID 21201, and "~Department of Radiology and Division of Comparative Medicine, Johns Hopkins Universily, MO Traylor, Baltimore, Maryland, U.S.A.
Summary This report describes five cases of myocarditis in the common cuttlefish, Sepia offcinalis. Both the systemic heart and the branchial hearts exhibited inflammatory lesions. Vibrio species were isolated from four of these cases.
Introduction In man and vertebrate animals, myocarditis is a lesion associated with many systemic diseases. The infectious causes ofmyocarditis include viruses, chlamydia, rickettsia, bacteria, fungi, protozoa and metazoa (Roberts, 1978; Kawai, Matsumori and Fujiwara, 1987; Robbins, Cotran and Kumar, 1984; Woodruff, 1980). Non-infectious causes of myocarditis include physical agents, such as radiation or heat, and hypersensitivity reactions to drugs (Robbins el al., 1984; Jubb, Kennedy and Palmer, 1985). Abnormal cardiac function and clinical signs may be completely absent in some cases of myocarditis (Ablemann, 1966; Lee, Char, Dianzumba and Prussia, 1986). In man, an estimated 1 to 4 per cent of routine necropsies exhibit some evidence of myocarditis (Robbins el al., 1984). The following report describes five cases of myocarditis in an invertebrate, the common cuttlefish, Sepia 'o~cinalis. Five out of seven cuttlefish submitted to the University of Maryland Department of Pathology from the National Aquarium in Baltimore in a 1-year period had myocarditis. These animals had been in the aquarium collection for periods ranging from 4 weeks to 1 year. Environmental problems in the holding tank were directly associated with mortality in four of the five myocarditis cases. These problems included copper exposure, temperature shock and high salinity shock. The circumstances in the fifth case were indicative of environmental problems, but unconfirmed.
Clinical History Two animals (1 and 2), whose deaths were associated with a 10-day exposure JPresent address: 4700 Hillsborough St., at William Moore Drive, Raleigh, NC 27606, USA. .T.Present address: U.S. Fish and Wildlife Service, 1825B Virginia Strect, Annapolis, MD 21401, U.S.A. 0021-9975[90/030291 + 07 $03.00/0
© 1990 Academic Press Limited
292
It. Reimschuessel
e t al.
to copper concentrations of 0"06 ppm, showed no premonitory clinical signs and were feeding actively 24 h before their deaths. They received no drug therapy. They were both males, 13"5 and 13 cm long, respectively. One animal, a 9-cm male (3), developed two 1-cm circular lesions in the epithelium and dermis of the mantle approximately 1 week before its death. This animal was not treated with antibiotics. It was somewhat more excitable than normal cuttlefish but was otherwise unremarkable and fed normally up to the day of its death. The possibility of a salinity accident was associated with this loss, but was never confirmed. The fourth cuttlefish, an l l - c m male (4), was clinically normal, eating actively and responsive to stimuli when a severe temperature shock following water changes contributed to the animal's demise. The fifth case (5) was a 15-cm male which had been in the collection for approximately 1 year. This animal had a chronic keratitis of the left eye manifested as an opaque white circular lesion in the centre of the cornea. This animal was placed on oral tetracycline at 10 mg per kg once a day in food as a treatment for suspected bacterial keratitis. After 5 days of this therapy, the animal's eye had not changed appreciably. An accident increasing the salinity to 41 ppt on the sixth day of therapy contributed to this animal's death. Pathology The animals were necropsied, and tissue samples were placed in 10 per cent phosphate-buffered formalin. Tissues were processed and embedded in paraffin wax and 6-p.m thick sections were stained with haematoxylin and eosin (HE) (Luna, 1968). Bacterial cultures were taken from lesions, haemolymph samples and swabs of the abdominal cavity. There were no gross lesions other than those noted clinically. Inflammatory loci were found in histological sections of both the systemic and branchial hearts of all but one of these cuttlefish. Other microscopic lesions included branchitis, inflammation of Needham's sac and vas deferens, dermatitis, inflammation of the cuttlebone, inflammation of the retro-orbital white gland, keratitis and inflammation of the ink sac (Table 1).
Table 1 Inflammatory lesions in five cuttlefish w i t h myocarditis Szte of inflammation Systcmlc h e a r t Branchial h e a r t Gills N e e d h a m s sac Vas defercns Skin Cuttlebone Retro-orbital g l a n d Cornea Ink sac
Animal number 1
2
3
4
5
+ + + +
+ +
+
+ +
+ + + +
+ +
+ + + + +
+ +
Myocarditis in Cuttlefish
293
The basic pattern of the myocardial lesions was similar in all cases. There were one or more inflammatory loci located in the myocardium of the systemic and branchial hearts (Figs 1 and 2). These were composed of heterophilis, some of which had an eosinophilic cytoplasm and a large round basophilic nuclei. Some of the nuclei of these cells were segmented. Rod-shaped bacteria were noted in three cases (1, 2 and 4) (Fig. 3). There was focal destruction of myocytes in these areas. In one case (5) the systemic heart had an extensive diffuse infiltrate of heterophils, almost replacing one wall of the ventricle (Fig. 4). Vibrio species were isolated from four of the five samples submitted (Table 2). Aeromonas and Pseudomonas species were isolated from one case. Discussion
The common cuttlefish is generally very adaptable to captivity compared with other cephalopods (Boletzky and Hanlon, 1983). It is, however, sensitive to major disturbances of its environment. The direct cause of death in most of these cases was an environmental shock. The myocardial lesions identified by histopathology were not associated with overt clinical signs. It is reasonable to assume that most of these fish would have survived for extended periods if they had not been subjected to unsuitable water conditions. Cuttlefish 5, which survived for a prolonged period in captivity without any observed signs of cardiac disease, had the most extensive lesions. This suggests the possibility of a slowly developing chronic myocarditis. If this animal had lived, the myocarditis may have eventually resulted in cardiac failure and death. The cuttlefish hearts are part of an essentially closed circulatory system (Barnes, 1974; Leake, 1975). The haemolymph flows from a single cephalic vein and paired posterior mantle veins into the paired vena cavae. It then flows through the renal appendages and into the branchial hearts. Some filtration occurs in the branchial appendages producing the urine in the renal sac (Schipp, Hohn and Sch~ifer, 1971; Schipp and Hevert, 1981). The haemolymph is pumped from the branchial heart, through the pericardial appendage and into the afferent branchial veins. It is oxygenated in the gills and then flows via the efferent branchial vein into the auricles of the systemic heart. From there, it is pumped into the anterior and posterior aortas and into the peripheral vasculature. In bacteraemia, all three hearts are vulnerable to bacterial seeding in the myocardium, which can then become a nidus of infection. Inflammation was noted in several organ systems in these cases (Table 1) making precise determination of the origin of the initial infection difficult. The reproductive system of the cuttlefish provides the most direct portal of entry for bacteria fi'om the water, opening directly to the environment. Four of the five cases reported here had concomitant infections of the reproductive system. The respiratory system could also be a portal of entry, with bacteria breaching the epithelial barrier of the gills. The pattern of inflammation observed in the three cases with branchitis, however, supports a haematogenous spread to the gills.
294
R. R e i m s c h u e s s e l e t a l .
Fig. 1.
Systemic heart with numerous heterophils infiltrating between tile myocytes, There is focal myocyte degeneration. HE x 120.
Fig, 2.
Branchial heart with a large focus of inflammation adjacent to the serosal surlhce. HE x 120.
Myocarditis
in Cuttlefish
295
Fig. 3.
Branchial heart. Higher magnification of the focus of inflammation in Fig. 2. Note the rod-shaped bacteria surrounded by heterophils i~ the centre of this lesion. H E × 3800.
Fig. 4.
Systemic heart. The ventrlcular wall on the left side of the thick arrow has an extensive infiltrate (arrow) of heterophils. At this low magnification, the infiltrate makes the myocardium appear darkened. Note also the heterophils adhering to the endocardium (thin arrow). HE × 30.
296
R. R e i m s c h u e s s e l e t al. Table 2 Bacterial isolates from cuttlefish with myoearditis
Animal
Specimen
Isolate
l 3
Abdominal fluid Haemolymph
4 5 5
Haemolymph Haemolymph Eye
Vibrioalginolyticus Aeromonashydrophila Pseudomonasputrefaciens Vibriosp, Vibrioalginolyticus Vibrioparahemolytieus
Vibrio spp. were isolated in four of the five samples examined (Table 2). Gram-variable rods were observed in some, but not all, cardiac lesions. Vibrio spp. are commonly cultured from aquatic organisms but their role in the pathogenesis of disease is not well understood (Roberts, 1978; Sparks, 1985). The Aeromonas and Pseudornonas organisms cultured from animal 3 were probably post-mortem invaders as the animal was moderately autolysed at post-mortem examination. Vibrio cultures were obtained from fresher specimens, but their role in this disease remains speculative.
Acknowledgments
The authors thank Christian Haul, Andrew Kane and Ann Muhvich for their assistance with the postmortem examinations and tissue processing and the National Aquarium in Baltimore which provided the specimens.
References
Ablemann, W. H. (1966), Myocarditis. New England Journal of Medicine, 275, 944-945. Barnes, R. D. (1974). Invertebrate Zoology. W. B. Saunders Company, Philadelphia. Boletzky, S. V. and Hanlon, R. T. (1983). A review of the laboratory maintenance, rearing and culture of cephalopod molluscs. In Memoirs of the National Museum of Victoria, No 44, Proceedings of the workshop on the biology and resource potential of cephalopods. D. M. Stone, Ed., Melbourne Australia. Jubb, K. V. F., Kennedy, P. C. and Palmer (1985). Pathology of Domestic Animals, Academic Press, New York. Kawai, C., Matsumori, A. and Fujiwara, H. (1987). Myocarditis and dilated cardiomyopathy. Animal Review of Medicine, 38, 221-239. Leake, L. D. (1975). Comparative Histology. Academic Press, New York. Lee, M. G., Char, G., Dianzumba, S. and Prussia, P. (1986). Cardiac involvement in severe leptospirosis. West Indian Medical Journal, 35, 295-300. Luna, L. G. (1968). Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology, 3rd edit. McGraw-Hill Book Company, New York. Robbins, S. L., Cotran, R. S. and Kumar, V. (1984). Pathologic Basis of Disease, 3rd edit. W. B. Saunders Company, Philadelphia. Roberts, R.J. (1978). Fish Pathology, Bailli~re Tindall Press, London. Schipp, R. and Hevert, F. (1981). Ultrafiltration in the branchial heart appendage of dibranchiate cephalopods; a comparative ultrastructural and physiological study. Journal of Experimental Biology, 92, 23-35.
Myocarditis in Cuttlefish
297
Schipp, R. Hohn, P. and SchS.fer, A. (1971). Elektronenmikroskopische und histochemische Untersuchungen zur Funktion des Kiemenherzanhanges (Pericardialdruse) yon Sepia oj]icinalis, geitschrift fiir gellforschung, 117, 252-274. Sparks, A. K. (1985). Synopis of Invertebrate Pathology. Elsevier Science Publishers B. V., New York. Woodruff, J. F. (1980) Viral myocarditis: A review. American Journal of Pathology, 101, 425-479.
[Receivedfor publication, April 5th 1989]
Myocarditis in the C o m m o n Cuttlefish
(Sepia oj~cinalis ) R. Reimschuessel*, M. K. Stoskopf] and R. O. Bennett+ *Department of Pathology, University of Maryland School of Medicine, 10 S. Pine Street, Baltimore, AID 21201, and "~Department of Radiology and Division of Comparative Medicine, Johns Hopkins Universily, MO Traylor, Baltimore, Maryland, U.S.A.
Summary This report describes five cases of myocarditis in the common cuttlefish, Sepia offcinalis. Both the systemic heart and the branchial hearts exhibited inflammatory lesions. Vibrio species were isolated from four of these cases.
Introduction In man and vertebrate animals, myocarditis is a lesion associated with many systemic diseases. The infectious causes ofmyocarditis include viruses, chlamydia, rickettsia, bacteria, fungi, protozoa and metazoa (Roberts, 1978; Kawai, Matsumori and Fujiwara, 1987; Robbins, Cotran and Kumar, 1984; Woodruff, 1980). Non-infectious causes of myocarditis include physical agents, such as radiation or heat, and hypersensitivity reactions to drugs (Robbins el al., 1984; Jubb, Kennedy and Palmer, 1985). Abnormal cardiac function and clinical signs may be completely absent in some cases of myocarditis (Ablemann, 1966; Lee, Char, Dianzumba and Prussia, 1986). In man, an estimated 1 to 4 per cent of routine necropsies exhibit some evidence of myocarditis (Robbins el al., 1984). The following report describes five cases of myocarditis in an invertebrate, the common cuttlefish, Sepia 'o~cinalis. Five out of seven cuttlefish submitted to the University of Maryland Department of Pathology from the National Aquarium in Baltimore in a 1-year period had myocarditis. These animals had been in the aquarium collection for periods ranging from 4 weeks to 1 year. Environmental problems in the holding tank were directly associated with mortality in four of the five myocarditis cases. These problems included copper exposure, temperature shock and high salinity shock. The circumstances in the fifth case were indicative of environmental problems, but unconfirmed.
Clinical History Two animals (1 and 2), whose deaths were associated with a 10-day exposure JPresent address: 4700 Hillsborough St., at William Moore Drive, Raleigh, NC 27606, USA. .T.Present address: U.S. Fish and Wildlife Service, 1825B Virginia Strect, Annapolis, MD 21401, U.S.A. 0021-9975[90/030291 + 07 $03.00/0
© 1990 Academic Press Limited
292
It. Reimschuessel
e t al.
to copper concentrations of 0"06 ppm, showed no premonitory clinical signs and were feeding actively 24 h before their deaths. They received no drug therapy. They were both males, 13"5 and 13 cm long, respectively. One animal, a 9-cm male (3), developed two 1-cm circular lesions in the epithelium and dermis of the mantle approximately 1 week before its death. This animal was not treated with antibiotics. It was somewhat more excitable than normal cuttlefish but was otherwise unremarkable and fed normally up to the day of its death. The possibility of a salinity accident was associated with this loss, but was never confirmed. The fourth cuttlefish, an l l - c m male (4), was clinically normal, eating actively and responsive to stimuli when a severe temperature shock following water changes contributed to the animal's demise. The fifth case (5) was a 15-cm male which had been in the collection for approximately 1 year. This animal had a chronic keratitis of the left eye manifested as an opaque white circular lesion in the centre of the cornea. This animal was placed on oral tetracycline at 10 mg per kg once a day in food as a treatment for suspected bacterial keratitis. After 5 days of this therapy, the animal's eye had not changed appreciably. An accident increasing the salinity to 41 ppt on the sixth day of therapy contributed to this animal's death. Pathology The animals were necropsied, and tissue samples were placed in 10 per cent phosphate-buffered formalin. Tissues were processed and embedded in paraffin wax and 6-p.m thick sections were stained with haematoxylin and eosin (HE) (Luna, 1968). Bacterial cultures were taken from lesions, haemolymph samples and swabs of the abdominal cavity. There were no gross lesions other than those noted clinically. Inflammatory loci were found in histological sections of both the systemic and branchial hearts of all but one of these cuttlefish. Other microscopic lesions included branchitis, inflammation of Needham's sac and vas deferens, dermatitis, inflammation of the cuttlebone, inflammation of the retro-orbital white gland, keratitis and inflammation of the ink sac (Table 1).
Table 1 Inflammatory lesions in five cuttlefish w i t h myocarditis Szte of inflammation Systcmlc h e a r t Branchial h e a r t Gills N e e d h a m s sac Vas defercns Skin Cuttlebone Retro-orbital g l a n d Cornea Ink sac
Animal number 1
2
3
4
5
+ + + +
+ +
+
+ +
+ + + +
+ +
+ + + + +
+ +
Myocarditis in Cuttlefish
293
The basic pattern of the myocardial lesions was similar in all cases. There were one or more inflammatory loci located in the myocardium of the systemic and branchial hearts (Figs 1 and 2). These were composed of heterophilis, some of which had an eosinophilic cytoplasm and a large round basophilic nuclei. Some of the nuclei of these cells were segmented. Rod-shaped bacteria were noted in three cases (1, 2 and 4) (Fig. 3). There was focal destruction of myocytes in these areas. In one case (5) the systemic heart had an extensive diffuse infiltrate of heterophils, almost replacing one wall of the ventricle (Fig. 4). Vibrio species were isolated from four of the five samples submitted (Table 2). Aeromonas and Pseudomonas species were isolated from one case. Discussion
The common cuttlefish is generally very adaptable to captivity compared with other cephalopods (Boletzky and Hanlon, 1983). It is, however, sensitive to major disturbances of its environment. The direct cause of death in most of these cases was an environmental shock. The myocardial lesions identified by histopathology were not associated with overt clinical signs. It is reasonable to assume that most of these fish would have survived for extended periods if they had not been subjected to unsuitable water conditions. Cuttlefish 5, which survived for a prolonged period in captivity without any observed signs of cardiac disease, had the most extensive lesions. This suggests the possibility of a slowly developing chronic myocarditis. If this animal had lived, the myocarditis may have eventually resulted in cardiac failure and death. The cuttlefish hearts are part of an essentially closed circulatory system (Barnes, 1974; Leake, 1975). The haemolymph flows from a single cephalic vein and paired posterior mantle veins into the paired vena cavae. It then flows through the renal appendages and into the branchial hearts. Some filtration occurs in the branchial appendages producing the urine in the renal sac (Schipp, Hohn and Sch~ifer, 1971; Schipp and Hevert, 1981). The haemolymph is pumped from the branchial heart, through the pericardial appendage and into the afferent branchial veins. It is oxygenated in the gills and then flows via the efferent branchial vein into the auricles of the systemic heart. From there, it is pumped into the anterior and posterior aortas and into the peripheral vasculature. In bacteraemia, all three hearts are vulnerable to bacterial seeding in the myocardium, which can then become a nidus of infection. Inflammation was noted in several organ systems in these cases (Table 1) making precise determination of the origin of the initial infection difficult. The reproductive system of the cuttlefish provides the most direct portal of entry for bacteria fi'om the water, opening directly to the environment. Four of the five cases reported here had concomitant infections of the reproductive system. The respiratory system could also be a portal of entry, with bacteria breaching the epithelial barrier of the gills. The pattern of inflammation observed in the three cases with branchitis, however, supports a haematogenous spread to the gills.
294
R. R e i m s c h u e s s e l e t a l .
Fig. 1.
Systemic heart with numerous heterophils infiltrating between tile myocytes, There is focal myocyte degeneration. HE x 120.
Fig, 2.
Branchial heart with a large focus of inflammation adjacent to the serosal surlhce. HE x 120.
Myocarditis
in Cuttlefish
295
Fig. 3.
Branchial heart. Higher magnification of the focus of inflammation in Fig. 2. Note the rod-shaped bacteria surrounded by heterophils i~ the centre of this lesion. H E × 3800.
Fig. 4.
Systemic heart. The ventrlcular wall on the left side of the thick arrow has an extensive infiltrate (arrow) of heterophils. At this low magnification, the infiltrate makes the myocardium appear darkened. Note also the heterophils adhering to the endocardium (thin arrow). HE × 30.
296
R. R e i m s c h u e s s e l e t al. Table 2 Bacterial isolates from cuttlefish with myoearditis
Animal
Specimen
Isolate
l 3
Abdominal fluid Haemolymph
4 5 5
Haemolymph Haemolymph Eye
Vibrioalginolyticus Aeromonashydrophila Pseudomonasputrefaciens Vibriosp, Vibrioalginolyticus Vibrioparahemolytieus
Vibrio spp. were isolated in four of the five samples examined (Table 2). Gram-variable rods were observed in some, but not all, cardiac lesions. Vibrio spp. are commonly cultured from aquatic organisms but their role in the pathogenesis of disease is not well understood (Roberts, 1978; Sparks, 1985). The Aeromonas and Pseudornonas organisms cultured from animal 3 were probably post-mortem invaders as the animal was moderately autolysed at post-mortem examination. Vibrio cultures were obtained from fresher specimens, but their role in this disease remains speculative.
Acknowledgments
The authors thank Christian Haul, Andrew Kane and Ann Muhvich for their assistance with the postmortem examinations and tissue processing and the National Aquarium in Baltimore which provided the specimens.
References
Ablemann, W. H. (1966), Myocarditis. New England Journal of Medicine, 275, 944-945. Barnes, R. D. (1974). Invertebrate Zoology. W. B. Saunders Company, Philadelphia. Boletzky, S. V. and Hanlon, R. T. (1983). A review of the laboratory maintenance, rearing and culture of cephalopod molluscs. In Memoirs of the National Museum of Victoria, No 44, Proceedings of the workshop on the biology and resource potential of cephalopods. D. M. Stone, Ed., Melbourne Australia. Jubb, K. V. F., Kennedy, P. C. and Palmer (1985). Pathology of Domestic Animals, Academic Press, New York. Kawai, C., Matsumori, A. and Fujiwara, H. (1987). Myocarditis and dilated cardiomyopathy. Animal Review of Medicine, 38, 221-239. Leake, L. D. (1975). Comparative Histology. Academic Press, New York. Lee, M. G., Char, G., Dianzumba, S. and Prussia, P. (1986). Cardiac involvement in severe leptospirosis. West Indian Medical Journal, 35, 295-300. Luna, L. G. (1968). Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology, 3rd edit. McGraw-Hill Book Company, New York. Robbins, S. L., Cotran, R. S. and Kumar, V. (1984). Pathologic Basis of Disease, 3rd edit. W. B. Saunders Company, Philadelphia. Roberts, R.J. (1978). Fish Pathology, Bailli~re Tindall Press, London. Schipp, R. and Hevert, F. (1981). Ultrafiltration in the branchial heart appendage of dibranchiate cephalopods; a comparative ultrastructural and physiological study. Journal of Experimental Biology, 92, 23-35.
Myocarditis in Cuttlefish
297
Schipp, R. Hohn, P. and SchS.fer, A. (1971). Elektronenmikroskopische und histochemische Untersuchungen zur Funktion des Kiemenherzanhanges (Pericardialdruse) yon Sepia oj]icinalis, geitschrift fiir gellforschung, 117, 252-274. Sparks, A. K. (1985). Synopis of Invertebrate Pathology. Elsevier Science Publishers B. V., New York. Woodruff, J. F. (1980) Viral myocarditis: A review. American Journal of Pathology, 101, 425-479.
[Receivedfor publication, April 5th 1989]