immunological and hematological parameters in captive harbor seals (phoca vitulina)

MARINE MAMMAL SCIENCE, 11(3):314-323 (July 1995) 0 1995 by the Society for Marine Mammalogy

IMMUNOLOGICAL AND HEMATOLOGICAL PARAMETERS IN CAPTIVE HARBOR

SEALS (PHOCA VITULINA)

JENS NIELSEN

State Veterinary Institute for Virus Research, Lindholm, DK 4771 Kalvehave, Denmark

AESSTRACT

Peripheral blood was collected from 13 captive seals (12 harbor seals and 1 gray seal) of various ages to study different immunological and hematological parameters. In vitro mitogenic reactivity of blood lymphocytes was measured by means of a microculture lymphocyte transformation test (LTT). After stimulation with different doses of the mitogens concanavalin A (Con A) and pokeweed mitogen (PWM), all examined seals showed significant proliferative responses to each mitogen. Furthermore, mitogenic reactivity significantly decreased with animal age suggesting this parameter of seal lymphocyte function is age-related. The present experiments support that the LTT is a suitable tool to monitor the functional capacity of seal lymphocytes. By means of the erythrocyte-rosette (E:- rosette) test it was demonstrated that a subpopulation of mononuclear seal bloosd leucocytes formed rosettes with sheep red blood cells (SRBC). This observation indicates that the phenomenon probably represents a marker for T lymphocytes in the seal as in several other mammalian species. Furthermore, the percentage of SRBC rosette-forming cells decreased with the age of the animals. Total blood leucocyte counts and differential leucocyte counts were determined by light microscopy. The number of leucocytes varied considerably among individual animals. Parallel to a significant decline of the percentage of lymphocytes with age, a corresponding increase in the percentage of neutrophils was demonstrated. In all of the seals, the percentages of monocytes and eosinophils were found to be low with only minor individual variation. The LTT and the E-rosette test are suggested as potential tools to elucidate immunological disorders in the seal.

Key words: harbor seal, Phoca vitulina, captivity, peripheral blood, mononuclear leucocytes, mitogenic reactivity, lymphocyte transformation test, E-rosettes, age, immunology, hematology.

In 1988 the seal plague killed approximately 18,000 seals in European seas (Dietz et al. 1989). A morbillivirus, denoted phocine distemper virus, was confirmed as the primary cause of the epizootic (for reviews, see Kennedy 1990; Osterhaus et al. 1990). The possible role of environmental factors such as overpopulation, weather conditions, or pollution in the occurrence and course

314

NIELSEN: IMMUNOLOGICAL AND HEMATOLOGICAL PARAMETERS 315

of the disease has been discussed (Dietz et al. 1989). Special attention was given to the possibility of a triggering role for certain bioaccumulative substances and the possible impairment of seal immune defenses. At present, no available data clearly indicate the involvement of such factors. The debate, however, revealed that only limited information about the seal immune system was available. In order to meet this situation, the present study was initiated.

The capability of lymphocytes to respond to stimulation with mitogens is widely used to evaluate the functional capacity of lymphocytes in a number of domestic animal species (Kristensen et al. 1982). Immunocompetent, well- functioning lymphocytes are crucial elements of the mammalian immune system. Stimulating lymphocytes with a spectrum of mitogens at different concentrations may provide a profile of lymphocyte responsiveness facilitating the possibility of detecting immunological disorders. Most frequently, mitogenic reactivity is monitored by the lymphocyte transformation test (LTT). The applicability of this test as a tool to monitor lymphocyte function in the seal was examined in the present study.

The ability to bind heterologous erythrocytes spontaneously and to form rosettes is a characteristic of T lymphocytes in a number of animal species, including the cat, cow, horse, swine, and sheep (for a review, see Kristensen et al. 1982). Populations of mononuclear seal blood leucocytes were examined for rosette-formation with sheep red blood cells (SRBC).

Blood leucocytic parameters of seals have been described in a number of reports (Geraci 1971, Greenwood et al. 197 1, Geraci and Engelhardt 1974, Geraci and Smith 1975, Hawkey 1975, Engelhardt 1979, McConnel and Vaughan 1983). The aim of the hematological examinations carried out in the present study was to elucidate in further detail the total leucocyte count values and the leucocyte proportions in peripheral blood from captive seals of various ages.

METHODS

Animals-Eleven harbor seals (Phoca vitulina) and one gray seal (Halichoerus grypus) kept in captivity for varying periods of time in the Seawater Aquarium in Esbjerg (Denmark) were examined in connection with the annual draining and cleaning of the seal exhibition tank. Four of the harbor seals (nos. 7 1, 76, 80a and 80b) and the gray seal (no. 78) were picked up as abandoned pups and held in captivity since then. Six harbor seals (nos. 87a, 87b, 493, 588, 589, 608) were born in the Seawater Aquarium. The age of the individual animals at the time of examination appears in Tables 1-3. In addition, a one- day-old premature harbor seal pup (born in the Seawater Aquarium approximately three months before term) with a body weight of 1.9 kg was examined. None of the animals showed clinical signs of disease at the time of examination. Seals nos. 493 and 608 had been vaccinated three times with an inactivated canine distemper vaccine (Kavak iD, Duphar Veterinary Ltd.), the last vacci- nation carried out two weeks prior to the examination of the animals. For the blood sampling, the seals were physically restrained on a wooden pad, using

316 MARINE MAMMAL SCIENCE, VOL. 11, NO. 3, 1995

capture nets. One sample of peripheral blood was drawn from veins in the pelvic flipper (Geraci 1971) of each individual animal into glass tubes containing EDTA as anticoagulant and stored overnight at 4°C before further processing. The duration of the blood sampling procedure ranged between 5 and 15-20 min for individual animals.

Cell culture medium-The cell culture medium used was RPM1 1640 Me- dium (Gibco, Europe) supplemented with 12% heat-inactivated (56°C for 30 min) fetal calf serum (FCS, Gibco, Europe), 3.5 PM 2-mercaptoethanol, 10 mM L-glutamine, neomycin (0.05 g/L), and streptomycin (0.1 g/L).

Mitogens-Stock solutions of the two plant lectins concanavalin A (Con A) grade III, (Sigma, U.S.A.) in sterile phosphate-buffered saline (PBS) and pokeweed mitogen (PWM) (Gibco, Europe) in sterile distilled water were stored at -20°C. Once thawed, they were immediately diluted with cell culture medium to the final concentrations.

Labelled precursor-[methyl-3H]-Thymidine (3H-Thymidine, specific activ- ity 2.0 Curies/mmols, 8.3 mCi/mg) obtained from Radiochemical Centre, Amersham, UK, was diluted with cell culture medium to a level of 40 pCi/mL.

Preparation of mononuclear leucocyte (MNL) suspensions-Isolation of MNL from stabilized blood was carried out by density gradient centrifugation essentially according to Boyum (1974). Briefly, blood was diluted with equal parts of sterile 0.85% sodium chloride. Volumes of approximately 5 mL of diluted blood were layered on top of 3 mL Lymphoprep (sodium metrizoate 9.6% (w/v) and 5.6% (w/v> Ficoll; density 1.077, Nyegaard & Co A/S, Norway) and centri- fuged at 800 g for 45 min at 4°C. The cells from the interphase were harvested and washed once in 0.83% ammonium chloride and twice in PBS (each centrifugation was at 160 g for 10 min at 4°C) before resuspending in cell culture medium. The cell suspensions were adjusted to 3 x lo6 cells/ml. Cells obtained from seal blood by the present isolation procedure were found to consist of more than 90% MNL, when examined on Maj-Grunwald-Giemsa stained cytocen- trifuge slides (J. Nielsen, personal observations). In the final cell suspensions, viability of cells was > 97% as determined by Trypan blue dye exclusion according to the method described by McLimans et al. (195 7).

Lymphocyte transformation test (LTT)--The LTT was carried out according to the microculture method used for pig lymphocyte stimulation assays in our laboratory (Nielsen 1987). Volumes of 100 PL of the MNL suspensions were distributed to individual wells of sterile round-bottomed 96-well microtiter plates (Nunc, Denmark), and volumes of 25 PL of the desired dilutions of Con A and PWM (as specified in Table 1) were added. All determinations. were performed in triplicate and included control cultures without mitogen. The plates were incubated at 37OC for 68 hours in a humidified atmosphere of 5% CO* and 95% air. Twenty hours before harvesting, 25 PL of the 3H-Thyrnidine dilution was added to each well. After incubation the cells were harvested and each sample was counted for 4 min in a Packard liquid scintillation spectro:meter. The incorporation of 3H-Thymidine was measured in counts per minute (cpm). Correction for quenching was omitted, since it remained constant within the experiments. For triplicate cultures, net counts per minute (Acpm) were deter-

Table 1. Mitogenic reactivity of peripheral blood lymphocytes from captive harbor seals to Con A and counts per minute (Acpm) of 3H-Thymidine incorporation.

Seal Age Con A pg/well

no’ (years) Sex 5 2.5 1.25 0.63 0.31 0.15 10 2.5

71 17.5 m* 72 17.5 f4 76 12.5 m 785 11.5 f 80a 8.5 f 80b 8.5 f 87a 1.5 m 87b 1.5 f

588 0.75 m 589 0.75 m 908 P’ m

1,0573 5,922 6,825 8,054 5,300 3,053 6,315 6,339 733 4,011 6,789 6,928 5,722 3,468 7,080 8,256

1,099 4,804 6,366 6,581 6,740 3,069 3,517 3,749 -350 3,123 6,265 11,157 9,969 8,582 5,352 6,066 2,748 14,255 19,35 1 18,099 11,791 5,932 7,539 7,458 3,706 18,200 25,692 27,904 22,267 14,2 10 9,086 13,177 4,095 34,151 47,654 59,888 35,363 24,035 15,920 20,404 3,304 22,075 34,03 1 46,301 35,556 10,313 17,180 19,467 4,966 30,391 49,736 40,3 12 33,276 27,163 nd6 nd 7,827 43,631 52,148 47,129 43,815 25,935 nd nd

11,763 11,851 9,255 2,586 2,491 1,745 nd 9,504 1 no = identification number; 2 m = m&; 3 = Acpm; 4 f = female; s 78 = gray sd, 6 nd = not done;


0 0.15 0.31 0.63 1.25 2.5 5.0

pg Con A/well

Figure 1. Con A dose-response profiles for blood lymphocytes from 0.75- to 1.5- yr-old seals (0) and 8.5- to 17.5-yr-old seals (0). Each point represents mean net counts per minute (Acpm) k standard error of the mean of four and six animals, respectively.

mined: Acpm = mean cpm of stimulated cultures minus mean cpm of control cultures.

Resetting procedzlre-Preparation of 2-amino-ethylisothiouroniumbromide (AET)-treated SRBC and the spontaneous rosetting procedure were performed according to the method routinely used in our laboratory for investigations in pigs (Nielsen 1986). Cells with three or more adherent red blood cells were considered as rosette forming. For each sample, at least 200 mononuclear cells were examined for erythrocyte-rosette (E-rosette) formation.

Total leucocyte counts--The total leucocyte count was carried out according to the standard method used in our laboratory: 25 /*L of EDTA-stabilized peripheral blood was mixed with 475 PLL methylrosaniline chloride (crystal violet) in glass tubes containing small glass balls. The stained cells were counted in a hemocytometer by light-microscopy and the total leucocyte number was cal- culated.

Differential 2e.wocyte cotints-Blood films prepared from the EDTA-stabilized samples were air-dried and stained with Azur-eosin-methylene blue solution (Giemsa’s solution, Merck Diagnostica, D-61 Darmstadt). For the differential counts, 200 leucocytes were identified by light-microscopy.

Statistical analysis-The data were analyzed using GraphPad In Stat v2.04a software (GraphPad Software, San Diego, CA, USA). Statistical significance was determined by t-test or Mann-Whitney Two Sample Test (M-W-test).

RESULTS

Blood from 11 of the 13 available seals was used for the LTT assay. All the seals examined showed strong proliferative lymphocyte responses upon mitogen


Table 2. Erythrocyte-rosette formation of peripheral blood mononuclear leucocytes from captive harbour seals.

Seal no’

71 72 76 785

80a 80b 87a 87b

Age (years)

17.5 17.5 12.5 11.5

8.5 8.5 1.5 1.5

Sex

m3 f4

T

f f 7

SRBC2 rosette-forming

cells (% of mononuclear

leucocytes)

26 28 20 17

:: 38 41

r no = identification number; 2 SRBC = sheep red blood cells; 3 m = male; 4 f = female; 5 78 = gray seal.

stimulation (Table 1). Individual maximum Acpm values expressed significant (t-test) mitogenic reactivity to Con A (P < 0.0007) and PWM (P < 0.002). In general, mitogenic reactivity to Con A was higher than to PWM. Using different concentrations of the two mitogens, the majority of the animals gave the highest response with 0.63 pg Con A/well and 2.5 pg PWM/well, respectively. The incorporation of 3H-Thymidine into stimulated cells was highest in the younger animals and mitogenic reactivity markedly declined with increasing animal age (Fig. 1). Thus, maximum responses of the group of 0.75-l. J-year- old seals were significantly (t-test) higher for Con A (P < 0.005) and PWM (P = 0.002) than maximum responses of the group of 8.5-17.5-year-old seals. The premature pup showed good reactivity to Con A, though lower than for the animals up to 8.5 years of age. Maximum 3H-Thymidine incorporation for this pup, however, was achieved with a higher mitogen concentration than for the other animals tested.

The incorporation of 3H-Thymidine into unstimulated control cultures ranged from 630 cpm (seal no. 76) to 2,341 cpm (seal no. 87a) for individual animals with an average of 1,333 cpm.

In all mononuclear cell suspensions examined, a subpopulation of cells (range 17%-41%) formed rosettes with SRBC (Table 2). Two of the seals (nos. 76 and 78) had markedly lower percentages of rosette-forming cells. However, the cell suspensions from these animals appeared with clusters of mononuclear cells which made the quantification difficult. The percentage of rosette-forming cells tended to decline with age and the recorded results revealed significantly fewer rosettes in 11.5-17.5-year-old seals than in 1.5-year-old seals (t-test, P = 0.01).

Total leucocyte counts (Table 3) varied considerably among individual animals. The lowest counts were found for the premature pup and in three older animals whereas the highest counts were recorded for the 0.75-year-old seals. Differential counts (Table 3) revealed that the percentage of neutrophils markedly


Table 3. Leucocytic parameters of peripheral blood samples from captive harbour seals.

Total counts Differential counts

Leuco- Lympho- Mono- Neutro- Eosino- Seal Age cytes cytes cytes phils phils no’ (years) Sex X 106/ml (%) (%I (%) (%I

71 17.5 m2 3.5 15 4 80 1 72 17.5 P 7.5 14 4 81 1 76 12.5 784 11.5 P

::; 16 2 81 1 :: 2

80a i:: : FE ;

:; 1 1

80b 17 1 87a 1.5 m 6:7 30

LZ 1

87b ;.:5

f 6.7 ;

57 1 493”

0:75 m 6.4 i: 35 2

588 m 7.7 34 ii

1 589 0.75 m 7.9 6

2: 1

608+ 0.75 f 8.5 ;; 2 62 4 908 P5 m 3.5 49 4 46 1

See Table 1 foomotes; + = vaccinated seals, see text. The differential counts were kindly confirmed by Per Henriksen, The National Veterinary Laboratory, Department of Diseases in Fish, Furbearing and Wild Animals, Aathus, Denmark.

increased with age while the percentage of lymphocytes declined. Thus, ncutro- phi1 counts were significantly lower (M-W-test, P = 0.001) and lymphocyte counts significantly higher (M-W-test, P = 0.001) for 0.75-1.5-year-old seals than for 8.5- 17.5-year-old seals, The percentages of monocytes and eosinophils were low and the minor individual variations could not be related to age. Seal no. 493 and the premature pup diverged from the other animals by showing higher percentages of lymphocytes and correspondingly lower percentages of neutrophils.

DISCUSSION

In accordance with previous reports (Leibold et al. 1989, De Swart et al. 1993), the present investigation demonstrated a strong proliferation of lymphocytes from captive harbor seals after stimulation with mitogens.

Generally, the individual dose-response values suggested that the mitogen doses used in the present study were suitable. However, the PWM dose-response values found in a few animals (nos. 71 and 80a) might indicate that peak stimulation had not been achieved.

The markedly decreased response demonstrated to both Con A and PWM, with growing age, suggests that the level of mitogenic reactivity in the seal is age-related. Furthermore, the present results support that the LTT is a suitable in vitro tool to monitor the functional capacity of lymphocytes in the seal as


suggested by Ross et al. (1993) and De Swart et al. (1993). Subsequently, the LTT has been used to demonstrate depressed mitogenic reactivity in harbor seals infected with canine distemper virus (J. Nielsen, personal observation), indicating this virus may cause immuno-suppression in harbor seals.

By means of the E-rosette test, a subpopulation of mononuclear seal blood leucocytes was demonstrated to form rosettes with SRBC. Although this phenomenon probably represents a marker for T lymphocytes in the seal as in several other mammalian species, experiments to confirm this asssumption have not been carried out. The decline in the percentage of E-rosettes observed with increasing age indicates that the level of rosette-forming cells in peripheral blood of healthy seals may be related to age.

The hematological findings of this study support and extend previous observations in captive harbor seals (Hawkey 1975, Engelhardt 1979, McConnel and Vaughan 1983). The total leucocyte counts of the seals examined in this study generally appeared to be low as compared to existing data. Whether this circumstance may be due to actual physiological fluctuations (e.g., stress) of the seals or attributed to artifactual differencies associated with techniques employed is not clear. Variations in leucocyte numbers may also occur because of patho- logical influences. Thus, leucopenia is often seen in animals suffering from disease, e.g., viral disease. However, since no signs of disease had either been recorded for the seals prior to blood sampling or in the following months, the observed low total leucocyte counts apparently did not reflect a state of disease. Rather low total leucocyte counts, as recorded for three of the older animals, have been described only occasionally. A possible explanation for this may be that previous studies almost exclusively refer to investigations on pups and younger animals.

The demonstrated decline of the percentage of lymphocytes with age and the corresponding increase of neutrophils strongly indicate that the age of the animal affects these parameters in healthy seals. In contrast, a similar relationship to age could not be demonstrated with regard to the recorded percentages of monocytes and eosinophils. The observed high percentage of lymphocytes in seal no. 493 may be explained by individual biological characters but might also express a state of immunological reactivity associated with the recent vac- cination of the animal. The almost equal distribution of neutrophils and lymphocytes observed for the premature pup most probably reflects a stage of ontogenetic development.

The immunological and hematological results for the gray seal (no. 78) showed only little variation as compared to the older harbor seals. However, the present study does not permit speculation on variations between different seal species.

The handling of the seals induced only minor signs of inconvenience to all animals, except one individual. Seal no. 87b appeared apathetic and exhibited signs of respiratory distress during blood sampling. However, the animal appeared to recover completely within a few hours, and a comparison of the results of the various tests for this seal with those of the age-matched seal (no. 87a) does not suggest that the handling had markedly influenced the examined parameters of seal no. 87b. Seal no. 80b gave birth to the premature pup (no.


908) approximately one month after the examination. However, whether this might be a result of unobserved stress in connection with the handling of the animal remains unanswered.

Unfortunately, it has not been posssible to carry out every test on each animal. The incompleteness of the data is due to the rather small volumes of blood which could be obtained from some of the animals by the blood sampling techniques used. Subsequent experience at our institute revealed that the ex- tradural technique described by Geraci and Smith (1975) is preferable when larger quantities of blood are required.

In conclusion, the present observations suggest that the L’IT and the E-rosette test are potential tools to elucidate immunological disorders in the seal.

ACKNOWLEDGMENTS

The cooperation of Svend Tougaard, Tyge Jensen and the technical staff at the Museum of Fisheries and Shipping, Seawater Aquarium, Esbjerg, is highly appreciated. Furthermore, I wish to thank Hanne Petersen, Hanne Egelund Hansen, and Bente Nilsson for technical assistance and Marianne Hansen for typing the manuscript.

LITERATURE CITED

BoYUM, A. 1974. Separation of blood leucocytes, granulocytes and lymphocytes. Tissue Antigens 4:269-274.

DE SWART, R. L., R. M. G. KLUTEN, C. J. HUIZING, L. J. VEDDER, P. J. H. REIJNDERS, I. K. G. VISSER, F. G. C. M. UYTDEHAAG AND A. D. M. E. OSTERHAUS. 1993. Mitogen and antigen induced B and T cell responses of peripheral blood mononuclear cells from the harbour seal (Phoca vitulina). Veterinary Immunology and lmmu- nopathology 37:217-230.

DIEtZ, R., M.-P. HEIDE-JORGENSEN AND T. HARKONEN. 1989. Mass deaths of harbor seals (Phoca vitulina) in Europe. Ambio 18:258-264.

ENGELHARDT, F. R. 1979. Haematology and plasma chemistry of captive pinnipeds and cetaceans. Aquatic Mammals 7:l l-24.

GERACI, J. R. 197 1. Functional hematology of the harp seal, Pagophilus groenlandicus. Physiological Zoology 44: 162-170.

GERACI, J. R., AND F. R. ENGELHARDT. 1974. The effects of storage time, temperature and anticoagulants on harp seal, Phoca groenlandica, hemograms: a simulated field study. Physiological Zoology 47~22-28.

GERACI, J. R., AND T. G. SMITH. 1975. Functional hematology of ringed seals (Phoca hispida) in the Canadian arctic. Journal of the Fisheries Research Board of Canada 32:.2559-2564.

I

GREENWOOD, A. G., S. H. RIDGWAY AND R. J. HARRISON. 1971. Blood values in young gray seals. Journal of the American Veterinary Medical Association 159:57 l- 574.

HAWKEY, C. M. 1975. Comparative mammalian haematology. William Heinemann Medical Books Ltd., London.

KENNEDY, S. 1990. A review of the 1988 European seal morbillivirus epizootic. Vet- erinary Record 1?7:563-567.

KriSTENSEN, F., B. KRISTENSEN AND S. LAZARY. 1982. The lymphocyte stimulation test in veterinary immunology. Pages 203-277 in F. Kristensen and D. F. Antczak, eds. Advances in veterinary immunology 1981. Elsevier, Amsterdam.

LEIBOLD, W., B. KUSZEWSKI, S. LUTKES, H. HEINZ, J. HEYMER, M. HADORN AND E.


DEEGEN. 1989. Hematological and immunological findings in seals. International workshop on current research in seal diseases. Hannover Veterinary School, February 21-22, 1989:21.

MCCONNEL, L. C., AND R. W. VAUGHAN. 1983. Some blood values in captive and free-living common seals (Phoca vitulina). Aquatic Mammals 10:9-13.

MCLIMANS, W. F., E. V. DAVIS, F. L. CLOVER AND G. W. RAKE. 1957. The submerged culture of mammalian cells: The spinner culture. Journal of Immunology 79:428- 433.

NIELSEN, J. 1986. Ontogeny of T cells in the pig fetus. Acta Veterinaria Scandinavica 27:343-350.

NIELSEN, J. 1987. Mitogenic reactivity of mononuclear cells isolated from thymus, spleen and umbilical cord blood of pig foetuses. Veterinary Immunology and Immuno- pathology 16:123-138.

OSTERHAUS, A. D. M. E., J. GROEN, H. E. M. SPIJKERS, H. W. J. BROEDERS, F. G. C. M. UYTDEHAAG, P. DE VRIES, J. S. TEPPEMA, I. K. G. VISSER, M. W. G. VAN DE BILDT AND E. J. VEDDER. 1990. Mass mortality in seals caused by a newly discovered virus-like morbillivirus. Veterinary Microbiology 23:343-350.

Ross, P. S., B. POHAJDAK, W. D. BOWEN AND R. F. ADDISON. 1993. Immune function in ftee-ranging harbour seal (Phoca vitulina) mothers and their pups during lactation. Journal of Wildlife Diseases 29:2 l-29.

Received: 18 November 1992 Accepted: 18 January 1995

immunological and hematological parameters in captive harbor seals (phoca vitulina)

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