- Email: [email protected]
Pathological findings indicative of distemper in European seals
Veterinary Microbiology, 23 (1990) 331-341 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
331
Pathological findings indicative of distemper in European seals A. Bergman 1, B. J~irplid 2 a n d B . - M . S v e n s s o n 1 ~Dept. of Pathology, Swedish University of Agricultural Sciences, Faculty of Veterinary Medicine, Box 7028, S- 75007 Uppsala (Sweden) 2Dept. of Pathology, National Veterinary Institute, Box 7073, S-750 07 Uppsala (Sweden)
ABSTRACT Bergman, A., J~irplid, B. and Svensson, B.-M., 1990. Pathological findings indicative of distemper in European seals. Vet. Microbiol., 23: 331-341. The first recorded cases of the recent epizootic were harbour seals observed at the Danish island of Anholt, 12 April 1988. The disease then spread throughout the sea waters of north-western Europe. The total mortality in Europe up to November 1988, was estimated to be at least 17 000 seals. The mortality rate in Danish-Swedish waters was about 60%. Autopsies including sampling for histology of most organs were performed on 37 harbour seals and 12 grey seals, collected mainly at the Swedish west coast and in the southern Baltic. In most of the harbour seals and in three of the grey seals we found histological changes in the upper and lower respiratory tracts, in the lower urinary tract and in the lymphatic system consistent with those diagnostic of distemper viral infection in the canine. These diagnostic criteria were: presence of intracytoplasmic eosinophilic inclusion bodies of epithelial cells of the trachea and the urinary bladder, interstitial pneumonia, and atrophy of lymphatic organs due to depletion oflymphocytes. Our findings in pathology of a canine distemper-like disease in the seals were presented in late August 1988, together with the Dutch findings in virology by Dr. Osterhaus and collaborators.
INTRODUCTION
Harbour seals (Phoca vitulina) affected with distemper were first observed at the Danish island o f Anholt in Kattegat, 12 April 1988. The disease then appeared throughout the sea waters o f north-western Europe with cases observed on the coasts of Schleswig-Holstein and the Netherlands in the early May and on the Swedish west cost from 19 May. The disease reached colonies o f harbour seals in the southern Baltic about one month later. Later on the disease also spread to harbour seals on the coasts of Norway and the British Isles. The disease caused mass mortality in affected harbour seal colonies. According to Dietz et al. ( 1989 ) the total mortality in Europe up to November 1988 was estimated to be at least 17 000 seals, with a mortality rate in Danish-Swedish waters of about 60%.
0378-1135/90/$03.50
© 1990 Elsevier Science Publishers B.V.
332
A. BERGMAN ET AL.
Besides harbour seals a limited number of grey seals (Halichoerus grypus) died from the disease, a few of which were found on the Swedish coasts. The efforts to find the cause of the disease resulted in two reports. According to the first (Osterhaus, 1988 ) the probable cause of the disease was thought to be a picornavirus or a herpesvirus, or the combined effects of the two viral agents. The present diagnosis of a canine distemper-like disease (Osterhaus and Vedder, 1988 ) was the result of a Swedish-Dutch collaboration, and was based upon our findings in pathology presented below. Changes similar to those present in canine distemper viral infection (CDV) were found in the upper and lower respiratory tracts, in the lower urinary tract and in the lymphatic system. The Swedish and Dutch results in pathology and virology were presented at press conferences in Sweden and the Netherlands, 29 August 1988. Cosby et al. ( 1988 ) tentatively named the present seal-derived morbillivirus as phocine distemper virus ( P D V ) , and found it to be more closely related to CDV antigenically than to any of the other three known morbilliviruses, i.e. measles (MV), peste des petits ruminants (PPRV) and rinderpest (RPV). Diseases in seals caused by CDV or other morbilliviruses were not reported before 1988. In an interview with "New Scientist" in September 1988 (No. 1631, p. 21 ), M.A. Grachev, Director of the Limnological Institute in Irkutsk, Siberia, mentioned CDV as the cause of a recent epizootic disease in Baikal seals (Phoca sibirica). The results of the Swedish-Dutch collaboration regarding the seal epizootic in Europe led in a u t u m n 1988 to invitations to one of us (A. Bergman ) and to Dr. Osterhaus by Director Grachev to visit the Limnological Institute in Irkutsk, for mutual exchange of experiences of the two epizootics. The disease in Baikal seals lasted from autumn 1987 to October 1988, causing the deaths of a few thousand animals from a total population of about 90 000. The diagnosis of CDV in Baikal seals was established by observing clinical signs, by using methods in pathology and serology and by the oligonucleotide probing technique. Recent results of collaborative Soviet-Dutch-Swedish studies (Grachev et al., 1989; Osterhaus et al., 1989) indicate that the European and the Baikal seals were infected by the same virus or by closely related viruses. MATERIAL AND METHODS
The seals were collected from 19 May 1988 to November 1988. Because of the high mortality rate in the disease, only selected seals were sent for autopsy to verify the spread of the epizootic. Autopsies were performed on 49 animals altogether, 37 of which were harbour seals and 12 grey seals. Twentyone of the harbour seals were collected from the Swedish west coast, five in Oresund and 11 in the Baltic. Two of the grey seals were collected in Oresund and the rest in the Baltic. Eleven of the 49 seals were found caught
PATHOLOGICAL FINDINGS INDICATIVE OF DISTEMPER IN EUROPEAN SEALS
333
and drowned in fishing gear. The rest were just found dead or were killed because of signs of disease. Of the harbour seals, 26 were males and 11 were females. The males were two aborted fetuses, seven newborns, eight juvenile-subadults, and nine adults. The female harbour seals were four newborns, four juvenile-subadults, and three adults. Of the grey seals six were males and six were females. Of the males two were juvenile-subadults and four were adults. The female grey seals were one juvenile-subadult and five adults. The group of adults, both harbour seals and grey seals, included some aged individuals. Age classification was performed according to size, weight and morphology of body and sex organs, tooth wear, etc. Age determination according to tooth section technique has not yet been completed. Organ tissues for histology were fixed in 10% neutral buffered formalin and embedded in paraffin. Sections, about five p m thick, were stained with haematoxylin and eosin. Various other techniques were used for specific purposes. Smears from the epithelium of the trachea and the urinary bladder were stained for inclusion bodies by the Shorr technique ( 1941 ) modified by Page and Green (1942). Besides conventional histological techniques a peroxidase anti-peroxidase technique (Sternberger et al., ! 970) was used for immunohistochemical demonstration of viral antigens in different tissues. Material was also saved for virological, bacteriological and chemical investigations. RESULTS
Clinical signs According to Swedish observations (T. H/irk6nen, personal communication, 1988 ) affected seals as a rule lacked normal escape reactions, were unable to dive and suffered from severe respiratory distress. In the terminal stages subcutaneous swellings were evident and were especially marked in the upper parts of the cervical and thoracic regions. Some animals had catarrhal symptoms such as rhinitis, conjunctivitis and diarrhoea. A few animals showed disturbances of the central nervous system.
Macroscopical findings Harbour seals. Among the aborted fetuses and newborns of the harbour seals autopsied ( 13 cases altogether) only two newborns showed changes characteristic of this disease. Most of the harbour seals in the other age groups showed characteristic lesions, mainly localized in the respiratory and lymphatic organs. Almost consistent findings were signs of p n e u m o n i a with secondary se-
334
A. B E R G M A N ET AL.
vere e m p h y s e m a of the lungs, the m e d i a s t i n u m and the subcutaneous tissues of the dorsal parts o f the neck and thoracic regions, and atrophy of the lymphatic organs. In some cases there were also signs o f conjunctivitis and catarrhal inflammation o f the upper airways.
Grey seals. Three of the grey seals, all females and judged to be older than 20 years, showed lesions characteristic o f this disease. Besides these regularly occurring lesions, seen in connection with the viral disease, one juvenile male harbour seal and one of the three grey seal females, m e n t i o n e d above, showed severe congestion of the small intestine due to volvulus of the mesentery.
Histological examination The histological evaluation of the material is still incomplete. The results presented here deal mainly with the respiratory and lymphatic systems.
Fig. 1. Lung of infected harbour seal. Interstitial inflammation with wide interalveolar septa containing mononuclear cells and occasional granulocytes. H&E, X 200. Fig. 2. Trachea of infected harbour seal. Catarrhal inflammation and intracytoplasmic inclusion bodies (arrows). H&E, X 800.
PATHOLOGICALFINDINGS INDICATIVE OF DISTEMPER IN EUROPEAN SEALS
335
Respiratory system. The predominant lesion in the lungs was an inflammatory reaction localized to the interstitium (Fig. 1 ). The interalveolar septa were widened as a consequence of oedema and accumulation of mononuclear cells, predominantly of histiocytic type. Single granulocytes were occasionally also seen in the septa. The alveoli were usually distended (emphysema) and empty. This alveolar emphysema had usually also led to an emphysema of the interstitial tissue of the lung. Besides the nonpurulent interstitial p n e u m o n i a several animals also showed purulent inflammation as a bronchopneumonia with more or less pronounced abscessations. In such cases also the trachea usually was the site of a catarrhal inflammation. In young harbour seals inflammatory reactions due to lung parasites were common. Eosinophilic intracytoplasmic inclusion bodies were often seen in the epithelial cells of the trachea (Fig. 2 ).
Lymphatic tissues. All lymphatic tissues were in general characterized by depletion of lymphocytes. In the thymus the cortex was the most seriously affected part. Normally (Fig. 3 ), the cortex has a dense population of lymphocytes while that of the medulla is more thin. This makes the border between the cortex and medulla rather sharp and distinct. In this disease (Fig. 4), the cortical lymphocytes were more or less depleted, and the cortex therefore appeared narrow and thin with a more indistinct cortico-medullary border. Severe depletion of cortical lymphocytes also led to a reversed relationship between cortex and medulla in respect of cellularity, a so-called cortical inversion (J~irplid, 1968 ). In some cases only a few scattered lymphocytes remained in the cortex. In such cases a rim of cells with an immature appearance sometimes appeared just beneath the capsule. In some areas of the cortex of such cases there were also aggregates of cells with a large foamy cytoplasm. Sometimes, the thymic medulla appeared atrophic and depleted of lymphocytes and the Hassall's corpuscles were degenerated and partly calcified (Fig. 4 ). In the lymph nodes the depletion of lymphocytes led to atrophy of the cortical and paracortical regions. The lymphocyte population was thin and the normally more concealed reticular cell stroma (Figs. 5 ) was apparent (Fig. 6). The sinuses of the nodes were in general dilated and filled with fluid, often containing free macrophages and other mononuclear cells. Sometimes the sinuses, especially of the medulla and of the paracortex, were filled with macrophages (histiocytosis). In the cortex there were no active lymphoid follicles. In some cases, rounded foci of an eosinophilic hyaline material appeared, representing germinal centres of exhausted follicles. Plasma cells and plasma cell precursors sometimes also appeared in both the cortex, the paracortex and in the medullary cords. In case of purulent inflammation of the lungs there were also a lot ofgranulocytes in the sinuses of the bronchial lymph nodes.
336
A. BERGMAN ET AL.
Fig. 3. Thymus of normal harbour seal. Lymphocytepopulation is dense in cortex (dark) and less dense in medulla (light). H&E, X80.
Fig. 4. Thymus of infectedharbour seal. Severe lymphocytedepletion. Degenerationof Hassall's corpusclesin medulla.Indistinct cortico-medullaryborder. H&E, X80. In the spleen the most prominent lesions were located in the white pulp. The lymphocytes of both the periarteriolar lymphocyte sheath (PALS), which is thymus dependent, and the lymphoid follicle, which is thymus independent, were more or less depleted. In cases of severe lymphocyte depletion there was no visible PALS and the follicle area was pale (Figs. 7 and 8). The red pulp of the spleen was generally characterized by hyperaemia and by an increased number of macrophages (histiocytosis). In some cases small accumulations of plasma cells and plasma cell precursors appeared, mostly in connection with the white pulp. The tonsils and the gut associated lymphatic tissue (GALT) were also depleted of lymphocytes. In general, the lymphatic tissue of the tonsils was thin with absence of lymphatic follicles. In the gut the depleted lymphatic nodules of the Peyers patches appeared as pale areas composed of a reticular cell stroma (Fig. 9 ). Other organs. In some cases there were intracytoplasmic eosinophilic inclu-
PATHOLOGICAL FINDINGS INDICATIVE OF DISTEMPER IN EUROPEAN SEALS
337
Fig. 5. Lymph node of normal harbour seal. Follicle with germinal centre in cortex (above). Dense population oflymphocytes in paracortex (arrow). H&E, X 80. Fig. 6. Lymph node of infected harbour seal. Lymphocyte depletion of cortex and paracortex. Dilation of subcapsular and intermediary sinuses. Exhausted follicles (arrows) in the cortex. H&E, X 80. sion bodies in the epithelial cells o f the urinary bladder. One harbour seal had signs of inflammation in the brain. Laboratory work using techniques in i m m u n o h i s t o c h e m i s t r y is in progress. Viral antigen, as demonstrated by the presence o f immunoperoxidase positive material, was found principally in epithelial cells o f the gastro-intestinal tract.
Miscellaneous lesions Besides macroscopical and microscopical changes characteristic of the present viral disease, some adult animals had chronic organ lesions o f different types not connected with this disease. Especially in adult grey seals, such lesions were c o m m o n and included periodontitis, intestinal ulcers, arteriosclerosis, nephropathy and adrenocortical hyperplasia.
338
A. BERGMAN ET AL.
Fig. 7. Spleen of normal harbour seal. Densely populated white pulp around a central artery. H&E, × 80. Fig. 8. Spleen of infected harbour seal. Severe lymphocyte depletion of white pulp (centre) around a central artery (arrow) and general histiocytosis of the surrounding red pulp. H&E,
x80.
Fig. 9. Ileum of infected harbour seal. Severe lymphocyte depletion of lymphatic nodules of a Peyer's patch. H&E, X 30.
PATHOLOGICALFINDINGS INDICATIVE OF DISTEMPER IN EUROPEAN SEALS
3 39
Other investigations Bacteriological investigation of the pneumonic lungs revealed different types of bacteria, e.g. Bordetella bronchiseptica, beta-haemolytic streptococci, Escherichia coli, and Klebsiella pneumoniae. These bacteria were sometimes also found in the spleen, indicating bacteriaemia. DISCUSSION
The lesions of the respiratory and lymphatic systems of the seals are similar to those of distemper viral infection in dogs. Also the inclusion bodies present in the cytoplasm of the epithelial cells of the trachea and the urinary bladder are similar to those diagnostic of distemper in the canine (Jones and Hunt, 1983; Dungworth, 1985 ). In the lungs there were no signs of the lesions typical of the acute phase of canine distemper, e.g. accumulations of syncytial cells in the alveoli. Instead, the interstitial pneumonia seemed to have persisted for some time, as had the purulent processes with abscessations. The alveoli were in general empty and distended with air, an alveolar emphysema, which had then extended into the interstitium as. an interstitial emphysema and further into the mediastinum, thus causing respiratory distresS. The emphysema had also passed forward through the thoracic inlet and into the subcutaneous tissues. All this emphysema had hampered the animals' ability to dive. In the lymphatic tissues both T- and B-cell areas were depleted of lymphocytes probably as a result oflymphocytolytic effect of the virus (Tizard, 1987 ). There were no signs of acute lymphocyte destruction, suggesting that the lesions had persisted fer some time. Signs of lymphatic tissue regeneration Were not'usually seen. The presence of a subcapsular rim of immature cells in the thymic cortex in some cases could, however, reflect an attempt at regeneration. Those chronic organ lesions which were not connected with the viral disease and which were found especially in adult grey seals, viz: periodontitiS, intestinal ulcer, arteriosclerosis, nCphropathy, and adrenocortical hyperplasia were consistent with those present in the serious disease complex in Baltic grey and ringed seals described by Bergman and Olsson (1985, 1989). One probable factor in this disease complex is an immune suppression due to a high body burden of organochlorine compounds, such as PCB and DDT, which, in its turn, is a.consequence of the severe pollution of the Baltic (Bergman and Olsson, 1985, 1989). The question whether the outbreak and course of the epizootic was influenced by environmental pollution or had some connection with the algae bloom in the area during late winter and spring 1988, is at present a subject for speculation. Regarding the pollution factor it must be stressed that the
340
A. B E R G M A N ET AL.
recent epizootic of a similar distemper-like disease in Baikal seals affected a population which had comparatively low tissue levels of organochlorines (Bobovnikova et al., 1989). Regarding the source of the outbreak of this epizootic both migrating marine mammals and terrestrial m a m m a l s and birds have been discussed as possible carriers of the viral infection. Of special interest in this connection is a retrospective serological survey showing that a morbillivirus closely related to canine distemper virus had been present in the populations of harp seals (Phoca groenlandica) and arctic ringed seals (Phoca hispida hispida) in Greenland since at least 1985 (Have et al., 1989). Another finding of great interest in this connection is the two cases of fatal morbillivirus infection in c o m m o n porpoises (Phoecoenaphoecoena) collected on the coast of Northern Ireland (Kennedy et al., 1988). ACKNOWLEDGEMENTS
The authors owe a dept of gratitude to many persons around the Swedish coasts for their tremendous enthusiasm and efforts in providing us with material for this investigation. Special mention should be made of Doctor Tero H~irk/Snen and his staff at the Tj~irn6 Marine Laboratorium at Str/Smstad and Professor Mats Olsson and staff at the Section of Vertebrate Zoology, Swedish Museum of Natural History, Stockholm. The authors also gratefully acknowledge the fruitful cooperation and discussion regarding virological aspects with Associate Professors Berndt Klingeborn and Gunnar Rockborn at the Department of Virology at the National Veterinary Institute in Uppsala, Sweden, and with Doctor Albertus Osterhaus at the National Institute of Public Health and Environmental Protection in Bilthoven, The Netherlands. This investigation was supported by grants from the National Swedish Environment Protection Board and the World Wildlife Fund.
REFERENCES Bergman, A. and Olsson, M., 1985. Pathology of Baltic grey seal and ringed seal females with special reference to adrenocortical hyperplasia: Is environmental pollution the cause of a widely distributed disease syndrome? Finnish Game Res., 44: 47-62. Bergman, A. and Olsson, M., 1989. Pathology of Baltic grey and ringed seal males. Report regarding animals sampled 1977-1985. In: A.V. Yablokov and M. Olsson (Editors), Influence of Human Activities on the Baltic Ecosystem. Proc. Soviet-Swedish Symposium: Effect of toxic substances on dynamics of seal populations. Moscow, USSR, 14-18 April 1986, Leningrad Gidrovmeteoizdat. Bobovnikova, T.I., Dibtseva, A.V., Malakhov, S.G., Siverina, A.A. and Yablokov, A.V., 1989. The accumulation of chlororganic pesticides and polychlorbiphenyls in the hydrobionts of Lake Baikal. In: A.V. Yablokov and M. Olsson (Editors), Influence of Human Activities on
PATHOLOGICALFINDINGS INDICATIVE OF DISTEMPER IN EUROPEAN SEALS
341
the Baltic Ecosystem. Proc. Soviet-Swedish Symposium: Effect of toxic substances on dynamics of seal populations. Moscow, USSR, 14-18 April, Leningrad Gidrovmeteoizdat. Cosby, S.L., McQuaid, S., Duffy, N., Lyons, C., Rima, B.K., Allan, G.M., McCullough, S.J., Kennedy, S., Smyth, J.A., McNeilly, F, Craig, C. and (}rvell, C., 1988. Scientific correspondence, Nature, 336:115-116. Dietz, R., Heide-J6rgensen, M.-P. and H~rk6nen, T., 1989. Mass deaths ofharbour seals (Phoca vitulina) in Europe. Ambio, 18: 258-264. Dungworth, D.L., 1985. The respiratory system. In: K.V.F. Jubb, P.C. Kennedy and N. Palmer (Editors), Pathology of Domestic Animals, 3rd Edn, Vol. 2. Academic press, San Diego, pp. 476-481. Grachev, M.A., Kumarev, V.P., Mamaev, L.V., Zorin, V.L., Baranova, L.V., Denikina, N.N., Belikov, S.I., Petrov, E.A., Kolesnik, V.S., Kolesnik, R.S., Dorofeev, V.M., Beim, A.M., Kudelin, V.N., Nagieva, F.G. and Sidorov, V.N., 1989. Distemper virus in Baikal seals. Nature, 338: 209. Have, P., Dietz, R. and Heide-J6rgensen, M.-P., 1989. Survey of antibodies against seal distemper virus in seals in Greenland. Report, International Workshop on Current Research in Seal Diseases, Hannover Veterinary School, 21-22 February 1989. J~irplid, B., 1968. Radiation induced asymmetry and lymphoma of thymus in mice. Acta Radiol., Suppl. No. 279. Jones, T.C. and Hunt, R.D., 1983. Veterinary Pathology, 5th Edn. Lea and Febiger, Philadelphia, PA, pp. 436-442. Kennedy, S., Smyth, J.A., Cush, P.F., McCullough, S.J., Allan, G.M. and McQuaid, S., 1988. Viral distemper now found in porpoises. Nature, 336: 21. Osterhaus, A.D.M.E., 1988. Seal death. Nature, 334:301-302. Osterhaus, A.D.M.E. and Vedder, E.J., 1988. Identification of virus causing recent seal deaths. Nature, 335: 20. Osterhaus, A.D.M.E., Groen, J., Uytdehaag, F.G.C.M., Visser, I.K.G., v.d. Bildt, M.W.G., Bergman, A. and Klingeborn, B., 1989. Distemper virus in Baikal seals. Nature, 338: 209210. Page, W.G. and Green, R.G., 1942. An improved diagnostic stain for distemper inclusions. Cornell Vet., 32: 265-268. Shorr, E., 1941. A new technic for staining vaginal smears: III. A single differential stain. Science, 94: 545-546. Sternberger, L.A., Hardy, P.H., Cuculis, J.J. and Meyer, H.G., 1970. The unlabelled antibodyenzyme method of immunohistochemistry. Preparation and properties of soluble antigenantibody complex (horseradish peroxidase anti-horseradish peroxidase) and its use in identification of spirochetes. J. Histochem. Cytochem., 18:315-333. Tizard, I., 1987. Veterinary Immunology, 3rd Edn. W.B. Saunders, Philadelphia, PA, pp. 366368.
331
Pathological findings indicative of distemper in European seals A. Bergman 1, B. J~irplid 2 a n d B . - M . S v e n s s o n 1 ~Dept. of Pathology, Swedish University of Agricultural Sciences, Faculty of Veterinary Medicine, Box 7028, S- 75007 Uppsala (Sweden) 2Dept. of Pathology, National Veterinary Institute, Box 7073, S-750 07 Uppsala (Sweden)
ABSTRACT Bergman, A., J~irplid, B. and Svensson, B.-M., 1990. Pathological findings indicative of distemper in European seals. Vet. Microbiol., 23: 331-341. The first recorded cases of the recent epizootic were harbour seals observed at the Danish island of Anholt, 12 April 1988. The disease then spread throughout the sea waters of north-western Europe. The total mortality in Europe up to November 1988, was estimated to be at least 17 000 seals. The mortality rate in Danish-Swedish waters was about 60%. Autopsies including sampling for histology of most organs were performed on 37 harbour seals and 12 grey seals, collected mainly at the Swedish west coast and in the southern Baltic. In most of the harbour seals and in three of the grey seals we found histological changes in the upper and lower respiratory tracts, in the lower urinary tract and in the lymphatic system consistent with those diagnostic of distemper viral infection in the canine. These diagnostic criteria were: presence of intracytoplasmic eosinophilic inclusion bodies of epithelial cells of the trachea and the urinary bladder, interstitial pneumonia, and atrophy of lymphatic organs due to depletion oflymphocytes. Our findings in pathology of a canine distemper-like disease in the seals were presented in late August 1988, together with the Dutch findings in virology by Dr. Osterhaus and collaborators.
INTRODUCTION
Harbour seals (Phoca vitulina) affected with distemper were first observed at the Danish island o f Anholt in Kattegat, 12 April 1988. The disease then appeared throughout the sea waters o f north-western Europe with cases observed on the coasts of Schleswig-Holstein and the Netherlands in the early May and on the Swedish west cost from 19 May. The disease reached colonies o f harbour seals in the southern Baltic about one month later. Later on the disease also spread to harbour seals on the coasts of Norway and the British Isles. The disease caused mass mortality in affected harbour seal colonies. According to Dietz et al. ( 1989 ) the total mortality in Europe up to November 1988 was estimated to be at least 17 000 seals, with a mortality rate in Danish-Swedish waters of about 60%.
0378-1135/90/$03.50
© 1990 Elsevier Science Publishers B.V.
332
A. BERGMAN ET AL.
Besides harbour seals a limited number of grey seals (Halichoerus grypus) died from the disease, a few of which were found on the Swedish coasts. The efforts to find the cause of the disease resulted in two reports. According to the first (Osterhaus, 1988 ) the probable cause of the disease was thought to be a picornavirus or a herpesvirus, or the combined effects of the two viral agents. The present diagnosis of a canine distemper-like disease (Osterhaus and Vedder, 1988 ) was the result of a Swedish-Dutch collaboration, and was based upon our findings in pathology presented below. Changes similar to those present in canine distemper viral infection (CDV) were found in the upper and lower respiratory tracts, in the lower urinary tract and in the lymphatic system. The Swedish and Dutch results in pathology and virology were presented at press conferences in Sweden and the Netherlands, 29 August 1988. Cosby et al. ( 1988 ) tentatively named the present seal-derived morbillivirus as phocine distemper virus ( P D V ) , and found it to be more closely related to CDV antigenically than to any of the other three known morbilliviruses, i.e. measles (MV), peste des petits ruminants (PPRV) and rinderpest (RPV). Diseases in seals caused by CDV or other morbilliviruses were not reported before 1988. In an interview with "New Scientist" in September 1988 (No. 1631, p. 21 ), M.A. Grachev, Director of the Limnological Institute in Irkutsk, Siberia, mentioned CDV as the cause of a recent epizootic disease in Baikal seals (Phoca sibirica). The results of the Swedish-Dutch collaboration regarding the seal epizootic in Europe led in a u t u m n 1988 to invitations to one of us (A. Bergman ) and to Dr. Osterhaus by Director Grachev to visit the Limnological Institute in Irkutsk, for mutual exchange of experiences of the two epizootics. The disease in Baikal seals lasted from autumn 1987 to October 1988, causing the deaths of a few thousand animals from a total population of about 90 000. The diagnosis of CDV in Baikal seals was established by observing clinical signs, by using methods in pathology and serology and by the oligonucleotide probing technique. Recent results of collaborative Soviet-Dutch-Swedish studies (Grachev et al., 1989; Osterhaus et al., 1989) indicate that the European and the Baikal seals were infected by the same virus or by closely related viruses. MATERIAL AND METHODS
The seals were collected from 19 May 1988 to November 1988. Because of the high mortality rate in the disease, only selected seals were sent for autopsy to verify the spread of the epizootic. Autopsies were performed on 49 animals altogether, 37 of which were harbour seals and 12 grey seals. Twentyone of the harbour seals were collected from the Swedish west coast, five in Oresund and 11 in the Baltic. Two of the grey seals were collected in Oresund and the rest in the Baltic. Eleven of the 49 seals were found caught
PATHOLOGICAL FINDINGS INDICATIVE OF DISTEMPER IN EUROPEAN SEALS
333
and drowned in fishing gear. The rest were just found dead or were killed because of signs of disease. Of the harbour seals, 26 were males and 11 were females. The males were two aborted fetuses, seven newborns, eight juvenile-subadults, and nine adults. The female harbour seals were four newborns, four juvenile-subadults, and three adults. Of the grey seals six were males and six were females. Of the males two were juvenile-subadults and four were adults. The female grey seals were one juvenile-subadult and five adults. The group of adults, both harbour seals and grey seals, included some aged individuals. Age classification was performed according to size, weight and morphology of body and sex organs, tooth wear, etc. Age determination according to tooth section technique has not yet been completed. Organ tissues for histology were fixed in 10% neutral buffered formalin and embedded in paraffin. Sections, about five p m thick, were stained with haematoxylin and eosin. Various other techniques were used for specific purposes. Smears from the epithelium of the trachea and the urinary bladder were stained for inclusion bodies by the Shorr technique ( 1941 ) modified by Page and Green (1942). Besides conventional histological techniques a peroxidase anti-peroxidase technique (Sternberger et al., ! 970) was used for immunohistochemical demonstration of viral antigens in different tissues. Material was also saved for virological, bacteriological and chemical investigations. RESULTS
Clinical signs According to Swedish observations (T. H/irk6nen, personal communication, 1988 ) affected seals as a rule lacked normal escape reactions, were unable to dive and suffered from severe respiratory distress. In the terminal stages subcutaneous swellings were evident and were especially marked in the upper parts of the cervical and thoracic regions. Some animals had catarrhal symptoms such as rhinitis, conjunctivitis and diarrhoea. A few animals showed disturbances of the central nervous system.
Macroscopical findings Harbour seals. Among the aborted fetuses and newborns of the harbour seals autopsied ( 13 cases altogether) only two newborns showed changes characteristic of this disease. Most of the harbour seals in the other age groups showed characteristic lesions, mainly localized in the respiratory and lymphatic organs. Almost consistent findings were signs of p n e u m o n i a with secondary se-
334
A. B E R G M A N ET AL.
vere e m p h y s e m a of the lungs, the m e d i a s t i n u m and the subcutaneous tissues of the dorsal parts o f the neck and thoracic regions, and atrophy of the lymphatic organs. In some cases there were also signs o f conjunctivitis and catarrhal inflammation o f the upper airways.
Grey seals. Three of the grey seals, all females and judged to be older than 20 years, showed lesions characteristic o f this disease. Besides these regularly occurring lesions, seen in connection with the viral disease, one juvenile male harbour seal and one of the three grey seal females, m e n t i o n e d above, showed severe congestion of the small intestine due to volvulus of the mesentery.
Histological examination The histological evaluation of the material is still incomplete. The results presented here deal mainly with the respiratory and lymphatic systems.
Fig. 1. Lung of infected harbour seal. Interstitial inflammation with wide interalveolar septa containing mononuclear cells and occasional granulocytes. H&E, X 200. Fig. 2. Trachea of infected harbour seal. Catarrhal inflammation and intracytoplasmic inclusion bodies (arrows). H&E, X 800.
PATHOLOGICALFINDINGS INDICATIVE OF DISTEMPER IN EUROPEAN SEALS
335
Respiratory system. The predominant lesion in the lungs was an inflammatory reaction localized to the interstitium (Fig. 1 ). The interalveolar septa were widened as a consequence of oedema and accumulation of mononuclear cells, predominantly of histiocytic type. Single granulocytes were occasionally also seen in the septa. The alveoli were usually distended (emphysema) and empty. This alveolar emphysema had usually also led to an emphysema of the interstitial tissue of the lung. Besides the nonpurulent interstitial p n e u m o n i a several animals also showed purulent inflammation as a bronchopneumonia with more or less pronounced abscessations. In such cases also the trachea usually was the site of a catarrhal inflammation. In young harbour seals inflammatory reactions due to lung parasites were common. Eosinophilic intracytoplasmic inclusion bodies were often seen in the epithelial cells of the trachea (Fig. 2 ).
Lymphatic tissues. All lymphatic tissues were in general characterized by depletion of lymphocytes. In the thymus the cortex was the most seriously affected part. Normally (Fig. 3 ), the cortex has a dense population of lymphocytes while that of the medulla is more thin. This makes the border between the cortex and medulla rather sharp and distinct. In this disease (Fig. 4), the cortical lymphocytes were more or less depleted, and the cortex therefore appeared narrow and thin with a more indistinct cortico-medullary border. Severe depletion of cortical lymphocytes also led to a reversed relationship between cortex and medulla in respect of cellularity, a so-called cortical inversion (J~irplid, 1968 ). In some cases only a few scattered lymphocytes remained in the cortex. In such cases a rim of cells with an immature appearance sometimes appeared just beneath the capsule. In some areas of the cortex of such cases there were also aggregates of cells with a large foamy cytoplasm. Sometimes, the thymic medulla appeared atrophic and depleted of lymphocytes and the Hassall's corpuscles were degenerated and partly calcified (Fig. 4 ). In the lymph nodes the depletion of lymphocytes led to atrophy of the cortical and paracortical regions. The lymphocyte population was thin and the normally more concealed reticular cell stroma (Figs. 5 ) was apparent (Fig. 6). The sinuses of the nodes were in general dilated and filled with fluid, often containing free macrophages and other mononuclear cells. Sometimes the sinuses, especially of the medulla and of the paracortex, were filled with macrophages (histiocytosis). In the cortex there were no active lymphoid follicles. In some cases, rounded foci of an eosinophilic hyaline material appeared, representing germinal centres of exhausted follicles. Plasma cells and plasma cell precursors sometimes also appeared in both the cortex, the paracortex and in the medullary cords. In case of purulent inflammation of the lungs there were also a lot ofgranulocytes in the sinuses of the bronchial lymph nodes.
336
A. BERGMAN ET AL.
Fig. 3. Thymus of normal harbour seal. Lymphocytepopulation is dense in cortex (dark) and less dense in medulla (light). H&E, X80.
Fig. 4. Thymus of infectedharbour seal. Severe lymphocytedepletion. Degenerationof Hassall's corpusclesin medulla.Indistinct cortico-medullaryborder. H&E, X80. In the spleen the most prominent lesions were located in the white pulp. The lymphocytes of both the periarteriolar lymphocyte sheath (PALS), which is thymus dependent, and the lymphoid follicle, which is thymus independent, were more or less depleted. In cases of severe lymphocyte depletion there was no visible PALS and the follicle area was pale (Figs. 7 and 8). The red pulp of the spleen was generally characterized by hyperaemia and by an increased number of macrophages (histiocytosis). In some cases small accumulations of plasma cells and plasma cell precursors appeared, mostly in connection with the white pulp. The tonsils and the gut associated lymphatic tissue (GALT) were also depleted of lymphocytes. In general, the lymphatic tissue of the tonsils was thin with absence of lymphatic follicles. In the gut the depleted lymphatic nodules of the Peyers patches appeared as pale areas composed of a reticular cell stroma (Fig. 9 ). Other organs. In some cases there were intracytoplasmic eosinophilic inclu-
PATHOLOGICAL FINDINGS INDICATIVE OF DISTEMPER IN EUROPEAN SEALS
337
Fig. 5. Lymph node of normal harbour seal. Follicle with germinal centre in cortex (above). Dense population oflymphocytes in paracortex (arrow). H&E, X 80. Fig. 6. Lymph node of infected harbour seal. Lymphocyte depletion of cortex and paracortex. Dilation of subcapsular and intermediary sinuses. Exhausted follicles (arrows) in the cortex. H&E, X 80. sion bodies in the epithelial cells o f the urinary bladder. One harbour seal had signs of inflammation in the brain. Laboratory work using techniques in i m m u n o h i s t o c h e m i s t r y is in progress. Viral antigen, as demonstrated by the presence o f immunoperoxidase positive material, was found principally in epithelial cells o f the gastro-intestinal tract.
Miscellaneous lesions Besides macroscopical and microscopical changes characteristic of the present viral disease, some adult animals had chronic organ lesions o f different types not connected with this disease. Especially in adult grey seals, such lesions were c o m m o n and included periodontitis, intestinal ulcers, arteriosclerosis, nephropathy and adrenocortical hyperplasia.
338
A. BERGMAN ET AL.
Fig. 7. Spleen of normal harbour seal. Densely populated white pulp around a central artery. H&E, × 80. Fig. 8. Spleen of infected harbour seal. Severe lymphocyte depletion of white pulp (centre) around a central artery (arrow) and general histiocytosis of the surrounding red pulp. H&E,
x80.
Fig. 9. Ileum of infected harbour seal. Severe lymphocyte depletion of lymphatic nodules of a Peyer's patch. H&E, X 30.
PATHOLOGICALFINDINGS INDICATIVE OF DISTEMPER IN EUROPEAN SEALS
3 39
Other investigations Bacteriological investigation of the pneumonic lungs revealed different types of bacteria, e.g. Bordetella bronchiseptica, beta-haemolytic streptococci, Escherichia coli, and Klebsiella pneumoniae. These bacteria were sometimes also found in the spleen, indicating bacteriaemia. DISCUSSION
The lesions of the respiratory and lymphatic systems of the seals are similar to those of distemper viral infection in dogs. Also the inclusion bodies present in the cytoplasm of the epithelial cells of the trachea and the urinary bladder are similar to those diagnostic of distemper in the canine (Jones and Hunt, 1983; Dungworth, 1985 ). In the lungs there were no signs of the lesions typical of the acute phase of canine distemper, e.g. accumulations of syncytial cells in the alveoli. Instead, the interstitial pneumonia seemed to have persisted for some time, as had the purulent processes with abscessations. The alveoli were in general empty and distended with air, an alveolar emphysema, which had then extended into the interstitium as. an interstitial emphysema and further into the mediastinum, thus causing respiratory distresS. The emphysema had also passed forward through the thoracic inlet and into the subcutaneous tissues. All this emphysema had hampered the animals' ability to dive. In the lymphatic tissues both T- and B-cell areas were depleted of lymphocytes probably as a result oflymphocytolytic effect of the virus (Tizard, 1987 ). There were no signs of acute lymphocyte destruction, suggesting that the lesions had persisted fer some time. Signs of lymphatic tissue regeneration Were not'usually seen. The presence of a subcapsular rim of immature cells in the thymic cortex in some cases could, however, reflect an attempt at regeneration. Those chronic organ lesions which were not connected with the viral disease and which were found especially in adult grey seals, viz: periodontitiS, intestinal ulcer, arteriosclerosis, nCphropathy, and adrenocortical hyperplasia were consistent with those present in the serious disease complex in Baltic grey and ringed seals described by Bergman and Olsson (1985, 1989). One probable factor in this disease complex is an immune suppression due to a high body burden of organochlorine compounds, such as PCB and DDT, which, in its turn, is a.consequence of the severe pollution of the Baltic (Bergman and Olsson, 1985, 1989). The question whether the outbreak and course of the epizootic was influenced by environmental pollution or had some connection with the algae bloom in the area during late winter and spring 1988, is at present a subject for speculation. Regarding the pollution factor it must be stressed that the
340
A. B E R G M A N ET AL.
recent epizootic of a similar distemper-like disease in Baikal seals affected a population which had comparatively low tissue levels of organochlorines (Bobovnikova et al., 1989). Regarding the source of the outbreak of this epizootic both migrating marine mammals and terrestrial m a m m a l s and birds have been discussed as possible carriers of the viral infection. Of special interest in this connection is a retrospective serological survey showing that a morbillivirus closely related to canine distemper virus had been present in the populations of harp seals (Phoca groenlandica) and arctic ringed seals (Phoca hispida hispida) in Greenland since at least 1985 (Have et al., 1989). Another finding of great interest in this connection is the two cases of fatal morbillivirus infection in c o m m o n porpoises (Phoecoenaphoecoena) collected on the coast of Northern Ireland (Kennedy et al., 1988). ACKNOWLEDGEMENTS
The authors owe a dept of gratitude to many persons around the Swedish coasts for their tremendous enthusiasm and efforts in providing us with material for this investigation. Special mention should be made of Doctor Tero H~irk/Snen and his staff at the Tj~irn6 Marine Laboratorium at Str/Smstad and Professor Mats Olsson and staff at the Section of Vertebrate Zoology, Swedish Museum of Natural History, Stockholm. The authors also gratefully acknowledge the fruitful cooperation and discussion regarding virological aspects with Associate Professors Berndt Klingeborn and Gunnar Rockborn at the Department of Virology at the National Veterinary Institute in Uppsala, Sweden, and with Doctor Albertus Osterhaus at the National Institute of Public Health and Environmental Protection in Bilthoven, The Netherlands. This investigation was supported by grants from the National Swedish Environment Protection Board and the World Wildlife Fund.
REFERENCES Bergman, A. and Olsson, M., 1985. Pathology of Baltic grey seal and ringed seal females with special reference to adrenocortical hyperplasia: Is environmental pollution the cause of a widely distributed disease syndrome? Finnish Game Res., 44: 47-62. Bergman, A. and Olsson, M., 1989. Pathology of Baltic grey and ringed seal males. Report regarding animals sampled 1977-1985. In: A.V. Yablokov and M. Olsson (Editors), Influence of Human Activities on the Baltic Ecosystem. Proc. Soviet-Swedish Symposium: Effect of toxic substances on dynamics of seal populations. Moscow, USSR, 14-18 April 1986, Leningrad Gidrovmeteoizdat. Bobovnikova, T.I., Dibtseva, A.V., Malakhov, S.G., Siverina, A.A. and Yablokov, A.V., 1989. The accumulation of chlororganic pesticides and polychlorbiphenyls in the hydrobionts of Lake Baikal. In: A.V. Yablokov and M. Olsson (Editors), Influence of Human Activities on
PATHOLOGICALFINDINGS INDICATIVE OF DISTEMPER IN EUROPEAN SEALS
341
the Baltic Ecosystem. Proc. Soviet-Swedish Symposium: Effect of toxic substances on dynamics of seal populations. Moscow, USSR, 14-18 April, Leningrad Gidrovmeteoizdat. Cosby, S.L., McQuaid, S., Duffy, N., Lyons, C., Rima, B.K., Allan, G.M., McCullough, S.J., Kennedy, S., Smyth, J.A., McNeilly, F, Craig, C. and (}rvell, C., 1988. Scientific correspondence, Nature, 336:115-116. Dietz, R., Heide-J6rgensen, M.-P. and H~rk6nen, T., 1989. Mass deaths ofharbour seals (Phoca vitulina) in Europe. Ambio, 18: 258-264. Dungworth, D.L., 1985. The respiratory system. In: K.V.F. Jubb, P.C. Kennedy and N. Palmer (Editors), Pathology of Domestic Animals, 3rd Edn, Vol. 2. Academic press, San Diego, pp. 476-481. Grachev, M.A., Kumarev, V.P., Mamaev, L.V., Zorin, V.L., Baranova, L.V., Denikina, N.N., Belikov, S.I., Petrov, E.A., Kolesnik, V.S., Kolesnik, R.S., Dorofeev, V.M., Beim, A.M., Kudelin, V.N., Nagieva, F.G. and Sidorov, V.N., 1989. Distemper virus in Baikal seals. Nature, 338: 209. Have, P., Dietz, R. and Heide-J6rgensen, M.-P., 1989. Survey of antibodies against seal distemper virus in seals in Greenland. Report, International Workshop on Current Research in Seal Diseases, Hannover Veterinary School, 21-22 February 1989. J~irplid, B., 1968. Radiation induced asymmetry and lymphoma of thymus in mice. Acta Radiol., Suppl. No. 279. Jones, T.C. and Hunt, R.D., 1983. Veterinary Pathology, 5th Edn. Lea and Febiger, Philadelphia, PA, pp. 436-442. Kennedy, S., Smyth, J.A., Cush, P.F., McCullough, S.J., Allan, G.M. and McQuaid, S., 1988. Viral distemper now found in porpoises. Nature, 336: 21. Osterhaus, A.D.M.E., 1988. Seal death. Nature, 334:301-302. Osterhaus, A.D.M.E. and Vedder, E.J., 1988. Identification of virus causing recent seal deaths. Nature, 335: 20. Osterhaus, A.D.M.E., Groen, J., Uytdehaag, F.G.C.M., Visser, I.K.G., v.d. Bildt, M.W.G., Bergman, A. and Klingeborn, B., 1989. Distemper virus in Baikal seals. Nature, 338: 209210. Page, W.G. and Green, R.G., 1942. An improved diagnostic stain for distemper inclusions. Cornell Vet., 32: 265-268. Shorr, E., 1941. A new technic for staining vaginal smears: III. A single differential stain. Science, 94: 545-546. Sternberger, L.A., Hardy, P.H., Cuculis, J.J. and Meyer, H.G., 1970. The unlabelled antibodyenzyme method of immunohistochemistry. Preparation and properties of soluble antigenantibody complex (horseradish peroxidase anti-horseradish peroxidase) and its use in identification of spirochetes. J. Histochem. Cytochem., 18:315-333. Tizard, I., 1987. Veterinary Immunology, 3rd Edn. W.B. Saunders, Philadelphia, PA, pp. 366368.