0012-4966/04/0304- © 2004 MAIK “Nauka /Interperiodica”0149

Doklady Biological Sciences, Vol. 395, 2004, pp. 149–153. Translated from Doklady Akademii Nauk, Vol. 395, No. 4, 2004, pp. 569–573.Original Russian Text Copyright © 2004 by Sokolova.

Ecological immunology as a new direction in sci-ence has appeared fairly recently. This scientific teach-ing encompasses the problems related to studying theeffect of environmental factors on the state of immunereactivity of the organism [1]. One of the main goals ofecological immunology is to study the general princi-ples of changes in immunological reactivity of theorganism in the course of adaptation of humans andanimals to new environmental conditions [2].

It is known that the immune system represents aphysiological mechanism that most rapidly and sensi-tively responds to any environmental changes. Thismechanism gradually undergoes improvement in thecourse of evolution and is present in the majority ofcontemporary living organisms [3]. In view of this, thestudy of shifts in immune indices in humans and ani-mals upon ecological changes is a very convenientexperimental and practical approach for determinationof ecological safety in any ecosystem.

Many researchers use these indices in cetaceans forstudying the ecological state of the World Ocean [4–6].

This works focuses on the changes in cell immunity(T- and B-lymphocytes) and biochemical indices in theBlack Sea bottlenose dolphin (

Tursiops truncatus

) inthe course of adaptation to living in captivity.

A large complex of biotic and abiotic factors ofhuman-modified environment has a detrimental effecton the immune system of adapting dolphins. Duringadaptation, a weakened immune system of dolphinscannot produce an adequate immune response; this, inturn, often leads to infectious diseases of various etiol-ogy (especially during the first months of staying incaptivity). Dolphins are infected with microflora of ter-restrial origin (transmitted from service staff or otherspecies of terrestrial animals), with which they onlyrarely could get in contact in natural environment.

Thus, dolphins are exposed to a mass-scale attack ofvarious pathogenic agents. This, in turn, results in a sig-nificant load on the immune system of dolphins, deplet-ing it and inducing destructive changes in lymphocyto-poietic organs [7].

The goal of this study was to detect some life-timechanges in the immune reactivity indices in the dol-phins

T. truncatus

at different stages of adaptation toliving in captivity. The determination of hematologicaland biochemical indices served as an additional esti-mate of the physiological state of animals.

This study was performed in 2001–2002 at UtrishMarine Station (Severtsov Institute of Ecology andEvolution, Russian Academy of Sciences) with 36 wildadult dolphins captured in the Black Sea. These animalswere then adapted to living in captivity at the marinestation. During the study, the animals were condition-ally divided into six groups, depending on the terms ofstaying in captivity. In total, we analyzed 40 blood sam-ples (

n

= 40).To characterize the peripheral blood lymphocytes of

bottlenose dolphins, we determined the followingparameters: (1) the total amount of mononuclear leuko-cytes in 1

µ

l of blood in Goryaev’s camera (thou-sand/

µ

l); (2) the viability of lymphocytes (by stainingwith 1% Trypane blue); (3) the quantitative composi-tion and the ratio between the immunocompetent cells(characterizing the T- and B-immune system state, i.e.,the absolute and relative T- and B-lymphocyte count).

This was the first study to apply the method of lym-phocyte subpopulation separation [8] for obtaining T-and B-lymphocyte fractions of the bottlenose dolphin,taking into account the species specificity of the studyobject and the availability of commercial preparations.To separate T/B subpopulations of lymphocytes of thedolphins

T. truncatus

, we considered possible to usespecific antisera to immunoglobulins (IgM and IgG) ofother anima species, namely, pig and cow (obtainedfrom Kovalenko RIEV). This change is the method wasbased on our assumption on phylogenetic kindredbetween the second-water cetaceans and terrestrialartiodactyl mammals [9, 10].

Some Immunological and Biochemical Indices of the Black Sea Bottlenose Dolphin (

Tursiops truncatus

)during Adaptation to the Captivity Conditions

O. V. Sokolova

Presented by Academician D.S. Pavlov November 5, 2003

Received November 12, 2003

Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Leninskii pr. 33,Moscow, 119071 Russia

GENERAL BIOLOGY

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SOKOLOVA

Biochemical and hematological indices were deter-mined using the common method used in clinical labo-ratory practice [11, 12].

It should be noted that we determined the activity of

γ

-glutamyltransferase (GT) of bottlenose dolphins forthe first time. In addition, the determination of GTactivity is the most sensitive and informative screeningtest for liver diseases, which is most preferable com-pared to determination of aminotransferases or alkalinephosphatase [13].

We observed distinctive cross-reaction between B-lymphocyte of

T. truncatus

and specific antisera to IgGand IgM of both pig and cow: 85–90% of total numberof bottlenose dolphin B-lymphocyte has reacted.

The test for viability, performed prior to obtainingT- and B-lymphocyte fractions, showed that 90–95% oflymphocytes were viable.

At early stages of adaptation (two to three weeks,group 1), bottlenose dolphins were characterized by afairly low level of immune reactivity, compared to theanimals that stayed in captivity more than one year(group 6). The bottlenose dolphins from group 1 werecharacterized by (1) a decreased total count of mononu-clear leukocytes in the peripheral blood; (2) significantprevalence of T-cells over B-cells in the T/B lympho-cyte populations (Table 1, Fig. 1); (3) the followingchanges in changes high ESR (2.0–58.0 m/h; on aver-age, 25.7

±

13.5 mm/h), moderate leukocytosis (7.40–13.70 thousand/

µ

l; on average, 11.65

±

1.67 thou-sand/

µ

l) with a left nuclear shift in leukoformula, and alow percentage of lymphocyte in leukocytogram (4.00–21.00%; on average, 9.50

±

4.48%) (Table 3, Fig. 3);(4) a high content in blood serum of total protein, albu-min, creatinine, aspartate aminotransferase, and glu-cose (Table 2, Fig. 2).

During adaptation (five to fourteen weeks, groups2–5), the cell immunity indices in bottlenose dolphinsgradually tended to increase. By week 14 of adaptation,the total count of mononuclear leukocytes in peripheralblood increased to 2.050

±

0.345 thousand/

µ

l, and theproportion of B-lymphocytes in the T/B ratio increasedto 15.57

±

3.07%. Hematological and biochemical indi-ces changed nonuniformly, with significant data scat-tering and great standard error of the mean (

m

).

Bottlenose dolphins from group 6 exhibited thegreatest indices of cell immunity. The hematologicalindices varied within normal range, which is consistentwith the data obtained by A.G. Misyur andL.N. Bogdanova [14]. Biochemical indices varied.

Table 1.

Absolute and relative composition of T/B lymphocyte subpopulations in peripheral blood of the Black Sea bottle-nose dolphin (

Tursiops truncatus

) at different terms of adaptation to living in captivity (

X

±

m

)

Terms of adaptation of dolphins

Total mononuclear leukocytes,thousand/

µ

l

T-lymphocytes +0-cells,

thousand/

µ

l

B-lymphocytes,thousand/

µ

lT-lymphocytes +

0-cells, % B-lymphocytes, %

Group 1 (2–3 weeks;

n

= 4)1.613

±

0.283 1.423

±

0.215 0.174

±

0.074 90.06

±

2.59 9.94

±

2.59

Group 2 (5–7 weeks;

n

= 12)1.561

±

0.186 1.359

±

0.156 0.180

±

0.029 88.97

±

1.04 11.03

±

1.04

Group 3 (8–9 weeks;

n

= 7)2.122

±

0.211 1.886

±

0.152 0.317

±

0.043 85.37

±

1.99 14.63

±

1.99

Group 4 (11–12 weeks;

n

= 8)1.484

±

0.255 1.258

±

0.217 0.195

±

0.043 87.48

±

1.38 12.52

±

0.24

Group 5 (13–14 weeks;

n

= 5)2.050

±

0.345 1.740

±

0.348 0.288

±

0.049 84.43

±

3.07 15.57

±

3.07

Group 6 (more than one year;

n

= 4)2.455

±

0.477 1.942

±

0.376 0.477

±

0.087 80.24

±

0.71 19.76

±

0.71

100%

80

60

40

20

0

1 2 3 4 5

1 2 3 4 5 6

Group

:

Fig. 1.

Ratio between the indices of absolute and relativecomposition of T/B lymphocyte subpopulations in periph-eral blood of the Black Sea bottlenose dolphin (

Tursiopstruncatus

) at different terms of adaptation to living in cap-tivity: (

1

) total count of mononuclear leukocytes (ths/

µ

l),(

2

) T-lymphocytes + 0-cells (ths/

µ

l), (

3

) B-lymphocytes(ths/

µ

l), (

4

) T-lymphocytes + 0-cells (%), (

5

) B-lympho-cytes (%).

DOKLADY BIOLOGICAL SCIENCES

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SOME IMMUNOLOGICAL AND BIOCHEMICAL INDICES OF THE BLACK SEA 151

In all groups, great data scattering (min–max) wasobserved. We assumed that this was due to irregularityof adaptation process in different individuals.

Thus, the method applied for the first time to esti-mate the ratio between T- and B-lymphocytes of bottle-nose dolphins—the fractionation of T- and B-lympho-cyte subpopulations [8]—may be used for the determi-nation of immune state (the lymphocyte component) ofbottlenose dolphins. Apparently, data obtained with theuse of antisera of the same animals species, the bottle-nose dolphin, will be the most reliable. It is noteworthythat this method of life-time immunologic diagnosticsallows the development of immune reactivity in dol-phins to be monitored in dynamics. In addition, the cellimmunity indices (total count of mononuclear leuko-cytes in peripheral blood and the ratio between the T-and B-lymphocyte subpopulations), together withhematological and biochemical indices, may be used todetermine the degree of adaptation of bottlenose dol-phins to living in captivity.

The results obtained in this study suggest that, atearly stages of adaptation of bottlenose dolphins to liv-ing in captivity, their protective and adaptive mecha-nisms of cell immunity are suppressed. As mentionedabove, in the first months of adaptation, the total lym-phocyte count in peripheral blood in bottlenose dol-phins decreases, with the B-cell component of theimmune system being affected most strongly.

Upon further adaptation to living in captivity, thetotal count of mononuclear leukocytes in peripheralblood and the proportion of B-cells in the T/B ratioincreased. Physiological parameters of blood remainedwithin normal range. In view of this, it can be con-cluded that adaptation of bottlenose dolphins to living

Table 2.

Biochemical indices of peripheral blood serum of the Black Sea bottlenose dolphin (

Tursiops truncatus

) at differentterms of adaptation to living in captivity (

X

±

m

)

Index

Terms of adaptation

2–3 weeks(

n

= 4)5–7 weeks

(

n

= 8)8–9 weeks

(

n

=5)11–12 weeks

(

n

= 6)13–14 weeks

(

n

=4)more than one

year (

n

= 3)

1. Total protein, g/l 81.30

±

4.75 77.67

±

3.05 72.99

±

1.61 70.34

±

2.35 78.85

±

1.81 72.60

±

0.78

2. Albumin, g/l 37.96

±

1.33 33.98

±

1.26 34.56

±

0.75 30.39

±

1.94 31.35

±

1.59 29.40

±

2.11

3. Urea nitrogen, mM 14.09

±

2.25 12.42

±

1.01 13.31

±

0.57 11.89

±

0.99 12.12

±

0.96 12.03

±

2.06

4. Creatinine,

µM 120.95 ± 4.17 120.43 ± 9.38 444.00 ± 368.42 85.35 ± 17.03 81.17 ± 15.26 109.67 ± 17.61

5. Cholesterol, µM 3.35 ± 2.41 4.35 ± 0.41 3.92 ± 0.54 4.05 ± 0.32 3.77 ± 0.28 4.37 ± 0.57

6. Bilirubin total, µM 9.30 ± 0.85 8.49 ± 1.08 7.97 ± 1.53 10.20 ± 2.19 7.95 ± 1.48 10.27 ± 1.12

7. Bilirubin direct, µM 1.80 ± 0.14 1.44 ± 0.28 1.30 ± 0.35 2.03 ± 0.72 1.90 ± 0.69 1.50 ± 0.46

8. Ca, mM 0.88 (n = 1) 1.45 ± 0.20 0.76 (n = 1) 1.39 ± 0.11 1.68 ± 0.49 1.35 ± 0.15

9. Mg, mM 0.87 (n = 1) 0.84 ± 0.09 0.58 (n = 1) 0.79 ± 0.13 0.95 ± 0.35 0.65 ± 0.01

10. Fe, µM 43.85 ± 3.75 36.61 ± 2.36 39.60 ± 8.70 28.83 ± 3.91 35.59 ± 5.70 28.87 ± 10.29

11. Pi, mM 1.75 (n = 1) 2.80 ± 0.34 5.30 (n = 1) 3.00 ± 0.34 3.17 ± 0.61 3.22 ± 0.68

12. [gamma]-Glutamyl-transferase, IU/l

18.50 ± 0.71 18.20 ± 0.81 17.07 ± 0.67 18.20 ± 1.22 20.60 ± 1.60 21.47 ± 2.02

13. Aspartate ami-notransferase, IU/l

78.75 ± 1.06 52.96 ± 6.10 45.60 ± 7.30 50.45 ± 10.94 59.25 ± 11.80 66.40 ± 7.47

14. Alanine aminotrans-ferase, IU/l

36.30 ± 10.61 25.67 ± 5.34 25.30 ± 8.07 21.65 ± 8.44 34.27 ± 20.08 47.60 ± 20.52

15. Glucose, mM 5.00 ± 2.00 4.81 ± 0.66 4.38 ± 1.60 4.17 ± 0.42 4.10 ± 0.50 4.47 ± 0.60

1 2 3 4 5 6

100%

80

60

40

20

01 3 5 7 9 11 13 15

Group:

Fig. 2. Biochemical indices of peripheral blood serum of theBlack Sea bottlenose dolphin (Tursiops truncatus) at differ-ent terms of adaptation to living in captivity: (1) total pro-tein (g/l), (2) albumin (g/l), (3) urea nitrogen (mM), (4) cre-atinine (µM), (5) cholesterol (µM), (6) total bilirubin (µM),(7) direct bilirubin (µM), (8) Ca (mM), (9) Mg (mM),(10) Fe (µM), (11) P (mM), (12) γ-glutamyltransferase(IU/l), (13) aspartate aminotransferase (IU/l), (14) alanineaminotransferase (IU/l), and (15) glucose (mM).

152

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SOKOLOVA

in captivity is a long-term and uneven process. At leastone year is required for the bottlenose dolphin T. trun-catus to adapt to a radically changed environment.

Note that the observed distinct cross-reactivity (by85–90%) between bottlenose dolphin lymphocytes andcow and pig antisera to IgG and IgM may be regardedan additional corroboration of a close phylogenetic kin-

dred between absolute hydrobionts and terrestrial artio-dactyl mammals.

The results of this study may be used for determina-tion of the degree of suppression of the immune reac-tivity of the bottlenose dolphin T. truncatus in naturalenvironment as a result of exposure to detrimental eco-logical factors of anthropogenic origin. In addition, thismay serve as an index reflecting the consequences ofanthropogenic press on other cetaceans, as well as anindicator of the ecological state of the World Ocean.

The study of the processes of immune and biochem-ical adaptation in the bottlenose dolphin facilitates theunderstanding the characteristics of evolutionarilydetermined changes in the immune system of toothedwhales. Furthermore, the knowledge of the dynamics ofthe immunological aspect of adaptation may be helpfulin revealing the reasons of immunodeficiency in ceta-ceans.

REFERENCES

1. Khaitov, R.M., Pinegin, B.V., and Istamov, Kh.I., Eko-logicheskaya immunologiya (Ecological Immunology),Moscow: Vses. Inst. Rybov. Okeanogr., 1995.

2. Shubik, V.M., Problemy ekologicheskoi immunologii(Problems of Ecological Immunology), Leningrad:Meditsyna, 1976.

3. Roitt, I., Brostoff, J., and Male, D., Immunology, Lon-don: Mosby, 1998. Translated under the title Immu-nologiya, Moscow: Mir, 2000.

Table 3. Hematological indices in the Black Sea bottlenose dolphin (Tursiops truncatus) at different terms of adaptation toliving in captivity (X ± m)

Terms ofadaptation

Ery

thro

cyte

s,m

ln/µ

l

Hem

oglo

bin,

g/l

ESR

,m

m/h

Leu

kocy

tes,

ths/

µl

Leukoformula, %

Neutrophils

Eos

inop

hils

Bas

ophi

ls

Mon

ocyt

es

Lym

phoc

ytes

Oth

er(h

istio

cyte

s)

juni

or

stab

segm

enta

l

Group 1 (2–3 weeks; n = 4)

3.64 ± 0.21

174.5 ± 1. 8

25.7 ± 13.5

11.65 ± 1.67

0.25 ± 0.29

3.25 ± 0.98

59.25 ± 2.96

19.50 ± 2.85

0 6.50 ± 1.10

9.50 ± 4.48

1.75 ± 0.29

Group 2 (5–7 weeks; n = 12)

4.02 ± 0.12

174.7 ±3.5

9.7 ± 2.2

9.64 ± 0.70

0 3.08 ± 0.81

55.92 ± 2.62

23.42 ± 2.24

0 4.33 ± 0.76

12.75 ± 2.33

0.50 ± 2.20

Group 3 (8–9 weeks; n = 7)

3.78 ± 0.13

165.6 ± 2.6

6.1 ± 1.1

10.81 ± 1.02

0 2.00 ± 0.69

47.71 ± 2.01

29.57 ± 0.78

0 2.43 ± 0.43

18.29 ± 2.48

0

Group 4 (11–12 weeks; n = 8)

3.65 ± 0.16

175.2 ± 5.7

12.3 ± 8.3

8.78 ± 1.10

0.62 ± 0.19

2.13 ± 0.51

56.50 ± 1.07

23.63 ± 0.73

0 2.75 ± 0.60

14.25 ± 1.75

0.12 ± 0.13

Group 5 (13–14 weeks; n = 5)

3.55 ± 0.19

174.8 ± 9.9

15.6 ± 13.8

13.31 ± 4.29

1.00 ± 0.35

2.40 ± 1.15

57.80 ± 5.09

20.80 ± 4.29

0 3.40 ± 0.76

14.40 ± 2.17

0.20 ± 0.22

Group 6 (more than one year;n = 5)

3.77 ± 0.16

178.7 ± 4.6

3.5 ± 2.9

8.72 ± 1.74

0.50 ± 0.33

1.50 ± 0.58

57.50 ± 1.10

17.75 ± 2.18

0 2.50 ± 1.00

20.25 ± 2.42

0

1 2 3 4 5 6

100%

80

60

40

20

01 3 5 7 9 11

Group:

Fig. 3. Hematological indices in the Black Sea bottlenosedolphin (Tursiops truncatus) at different terms of adaptationto living in captivity: (1) erythrocytes (mln/µl), (2) hemo-globin (g/l), (3) ESR (mm/h), (4) leukocytes (ths/µl),(5) junior neutrophils, (6) stab neutrophils, (7) segmentalneutrophils, (8) eosinophils, (9) basophils, (10) monocytes,(11) lymphocytes, and (12) other cells.

DOKLADY BIOLOGICAL SCIENCES Vol. 395 2004

SOME IMMUNOLOGICAL AND BIOCHEMICAL INDICES OF THE BLACK SEA 153

4. Fournier, M., Degas, V., Colborn, T., et al., Toxicol. Lett.,2000, vol. 112/113, pp. 311–317.

5. Lahvis, G.P., Wells, R.S., Kuehl, D.W., et al., Environ.Health Persp., 1995, vol. 103, no. 4, pp. 67–72.

6. Lapierre, P., de Guise, S., Muir, D.C.G., et al., Environ.Res., Sect. A, 1999, vol. 80, pp. 104–112.

7. Romanov, V.V., The Immune Status of Bottle-nosed Dol-phins Kept in Captivity as a Criterion of Infection Resis-tance, Cand. Sci. (Biol.) Dissertation, Moscow, 1991.

8. Ernst, L.K., Shishkov, V.P., Orlova, A.R., et al., Moleku-lyarno-geneticheskie i statisticheskie metody izucheniyaglavnogo kompleksa gistosovmestimosti krupnogorogatogo skota v svyazi s ustoichivost’yu i vospriimchi-vost’yu k leikozam (metodicheskie rekomendatsii) (Pri-oritetnye napravleniya genetiki. PodprogrammaFTsNTP) (Molecular Genetic and Statistical Methods ofStudying the Main Histocompatibility Complex of Cat-tle in Relation to the Resistance and Susceptibility toLeukemia: A Manual (State Science and TechnologyProgram “Frontiers in Genetcs”)), Moscow: Inst.Obshch. Genet., 1998.

9. Yablokov, A.V., Bel’kovich, V.M., and Borisov, V.I., Kityi del’finy (Whales and Dolphins), Moscow: Nauka,1972.

10. Milinkovitch, M.C., Orti, G., and Meyer, A., Nature,1993, vol. 361, pp. 346–348.

11. Simonyan, G.A. and Khisamutdinov, F.F., Veterinarnayagematologiya (Veterinary Hematology), Moscow: Kolos,1995.

12. Men’shikov, V.V., Delektorskaya, L.N., Zolot-nitskaya, R.P., et al., Laboratornye metody issledovaniyav klinike. Spravochnik (Laboratory Methods in ClinicalPractice: A Reference Book), Moscow: Meditsyna,1987.

13. Lifshits, V.M. and Sidel’nikova, V.I., Biokhimicheskieanalizy v klinike. Spravochnik (Biochemical Tests inClinic: A Reference Book), Moscow: Triada-X, 2002.

14. Misyura, A.G. and Bogdanova, L.N., in Chernomor-skaya afalina Tursiops truncatus ponticus: morfologiya,fiziologiya, akustika, gidrodinamika (The Black SeaBottle-nosed Dolphin Tursiops truncatus ponticus: Mor-phology, Physiology, Acoustics, and Hydrodynamics),Moscow: Nauka, 1997, pp. 186–213.