On the Serological Constitution of Striped Dolphin(Prodelphinus caeruleo-albus (Meyen)) (I)

BY
KATSUMI y AMAGUCHI and KAZUO FUJINO(Received July 10, 1952)
TABLE OF CONTENTS
Chapter I. Introduction .................................................... 69
Chapter 2. Materials and Methods of the Experiment ...................... 70
Chapter 3. Isohaemagglutination ........................................... 71
Chapter 4. Serum-type and the Existence of Anti C Agglutinin in Serum .. 72
Chapter 5. Antigens Dc1 and Dc 2 Proved by An Immune Antibody . . . . . . . . 73
Paragraph 1. Anti Dc1 Serum and Anti Dc 2 Serum .................... 73
Paragraph 2. Agglutinin Value and Haemolysin Value ................. 73
Paragraph 3. Appearance Rate of Each Type .......................... 74
Chapter 6. Anti Dc1 and Anti Dc 2 Isohaemagglutinin Seen in Normal
Sera of Striped Dolphin ........................................ 75
Chapter 7. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Chapter 1. Introduction

Since Karl Landsteiner classified the human blood type according
to isohaemagglutination at the beginning of the 20th century, theclassification of blood type in various animals has been made according
to isohaemagglutination,1) 2) 3) 4 ) 5 ) isohaemolysis and immune antibody. 6 ) 7 )On the other hand, the advancement of serology has been so remarkable during the latest half century that its range covers the A, B, c,s) 9)0, M, N of blood corpuscles in various animals and the distributive stateof partial antigens 10)n)i2 ) in each character, up to the systematic evolution of animals and the indexing of heterotype antigens, and even to
the analysis of the structure of each formal substance.
Serological study on whales which belong to the aquatic Mammalia
has been hardly done up to the present. The authors, following the above
stated results brought by the forerun researchers, have discovered thetwo antigens, namely D 1 and D 2 , from the immune agglutinin andhaemolysine produced by immunizing rabbits with the blood corpusclesof the striped dolphin, one of the aquatic mammals, and, by it, havebeen able to classify the blood corpuscle of striped dolphin into threekinds. So it is a great honor for them to report on it so as to receive
a lot of precious critics from various worlds.

10

K. y AMAGUCHI and K. FUJINO

Chapter 2. Materials and Methods of the Experiment

Materials and methods of the experiment are to be summerized
in the following. Details will be given in each clause.

Blood Corpuscle and Serum of Striped Dolphin
The spouting blood was taken into a pot when a dolphin had beenpulled up to the operating place and his heart was stabbed with aknife. Blood corpuscles were separated from the blood clot on theabsorbent cotton after coagulation by using physiological saline solution.
After several times of centrifugal washing, the obtained blood corpusc.les
were used for immunization, adsorption and reaction test. The separated
serum was made inactive in the warm bath of 56°C for 30 minutes.
And then the physiological saline solution with 5% carbolic acid wasadded to it. (Its quantity was 1/10 of that of the serum). Afterenough mixing, it has been preserved in the ice room.
Human Blood Corpuscle
A part of the blood taken, for the test of Wassermann's reaction,from the elbow veine of a healthy person was washed several times
with physiological saline solution and was centrifuged. The precipitatedblood corpuscles obtained thu8 were used for adsorption and coagulating
reaction test.
Immune Animal
Serum type in the normal sera and whether the A character inthe saliva was discharging type or non-discharging type, namely A+
type or A-type, were examined in a healthy rabbit, 2.5 to 3.0 kg in
weight.
Immunizing Method
The blood corpuscles were washed several times with physiblogicalsaline water, and then the 10% floating liquid was made with saline
water. Each 5 cc of the liquid was injected into the ear veine of a
rabbit each other day. The total number of injection was 7.
Collecting and Preserving Method of Antiserum
One week after the last injection the whole blood was collected.
The separated serum was made inactive in the warm bath of 56°C for
30 minutes. And then the physiological saline solution with 5% carbolic
acid was added to it. (Its quantity was 1/10 of that of the serum).
After adequate mixing, it has been preserved in the ice room. Any
food had been given to the immune animal for about 12 hours before
the blood collecting so as to prevent the turbidity of serum.

Serological Constitution of Striped Dolphin

71

Testing Method of Coagulating Reaction and Hemolysis Reaction
30 minutes after, the coagulating reaction was judged by theholeglass method in the room temperature. The hemolysis reaction
was judged by the test tube method, adding marmot serum as complement, after 30 minutes warm bathing of 37°C.
Adsorption Test
For adsorption, appropriate amount of the washed precipitatedblood corpuscles had been mixed according to the dilution of antiserum.
After being left in the room temperature for a few hours, the upper
clear part was used for reaction.
Chapter 3.

lsohaemagglutination

Isohaemagglutination in striped dolphin is generally weak. On
rare occasions, however, it is comparatively strong, but not enough toTable 1. lsohaemagglutination in Prodelphinus caeruleo-albus Meyen~;~~~scle·ll

1 2 3 4 5 6 7 8 9 10 1112 13 14 15 1617 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 3~ 36

Serum
2
3
4
5
6
7

~

:.a0..
0"'"'·;:::
(!)

0..

...,
r/l

.....0

s::I
'"
(!)

r/l

OJ

s
z'"0

Blood corpuscle of striped dolphin

9
10
11
12
13
14
15
16
17

----m---H-Hm---rn----rn--------------------+-+-------+-------+----+-------------rn-H------------------H------H

l8

19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36

-------------~------------,---------

K. YAMAGUCHI and K. FUJINO

72

classify easily the blood type with it. As shown in the Table 1, thereaction to the Sera No. 4 and No. 16 is comparatively strong, andmoreover each agglutinin is adsorbed completely by each blood corpuscle
which reacts positively to it so that the relation to the immune agglutinin which is to be stated after is noticed. On the other hand, theSerum No. 6 reacts weakly, but it seems not to be connected withwhat is to be stated after.
Chapter 4. Serum-type and the Existence ofAnti C Agglutinin in Serum

Whether the agglutinins to the Human A, B and C characters
exist or not in the normal serum of striped dolphin was examined by
the coagulating reaction to each type of human blood. Some results of
the reaction are given in the Table 2.
Table 2.

Absorption. test of striped dolphin's normal serumby Human Blood Corpuscles A, B and 0

1 1 ++~Serum l,__ _ _ _ _ _ _ _s_e_ru_m_o_f_s_t_r_ip_e_d_d_o_lp_h_in_ _ _ _ _ _ _ __*~ ~Q)

6. i::

!; !;

A

0 00

B

l::Q ~

0h5]~

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

tfr -- +tt - - - - ++ - + ttt - + - - + - ++ - - - + - ++ ++ - - - ++ - - ttt -- ++ - --T-- +tt - +t - -++-----

--* -

+

- + - -

0

A
B
0

A

-~!~-!----------------------~-----

A

B

W----++-+ - - - - - + - - - - - -

B
0

*
**

Work human blood corpuscle
Absorption human blood corpuscle

As clarified in the above table, the Type a' is seen in the four
dolphins (16%), namely Nos. 1, 5, 14 and 20, while the Type O' in all
the rest, that is 21 dolphins (84% ). The Types (3' and a' (3' did notappear in any of the 25 dolphins. In the Type a', Nos. 1 and 5 react

'13

Sero1oglca1 Constitution of Striped :bo1phln

positively until the dilution with the water whose quantity is threetimes as much as the original liquid, and Nos. 13 and 19 react positively
only in the state of the original liquid. The existence of the normal
anti C agglutinin was not testified in ·any serum.
Chapter 5.

Paragraph 1.

Antigens Dc1 and Dc2 Proved by
An Immune Antibody
Anti Dc1 Serum and Anti Dc2 Serum

When a rabbit is immunized using as antigen the striped dolphin's
blood corpuscle which belongs to Dc1 , the agglutinin and haemolysin,
particular in each species, to the striped dolphin's blood corpuscle areproduced in the serum of the rabbit. Simultaneously anti Dc1 agglutinin
and haemolysin are also produced. From thus obtained antibodies theanti Dc1 immune agglutinin and haemolysin are obtained if the agglutinin
and haemolysin, particular in each species, are adsorbed away by Dc2
blood corpuscle. Anti Dc2 immune agglutinin and haemolysin are obtained by the same operation. By the immune serum obtained by theabove method, it was proved that the existences of both agglutinins
and the both haemolysins, namely Dc1 and Dc2 were perfectly consistenteach other.
Paragraph 2.

Agglutinin Value and Haemolysin Value

Examples of the agglutinin value and haemolysine value of theanti Dc1 and Dc2 immune sera obtained by the method given in theprevious Paragraph are shown in the Tables 3 and 4 respectively.
Table 3. Agglutinin value of anti Der and Dc2 immune
sera to each type of serum of striped dolphin
Anti Dc1 agglutinin value
Immune rabbit
I
Blood-- - .
Absorption Work
type of\
Exrste;nce or
blood \ blood
Serum Serum :non-eiostence corpuscle corpuscle striped
dolphinNo. j type of Character
A in saliva
1

No. 3
No. 7
Sf-

\'>'

01

I

I

I

I

No. 8
Dc 2

Der Dc2

No. 1

Der

No. 4

Dc2

Dilution of antiserum

1
20

1
40

1
1
1
1
80 160 320 640-ltt

ttt I ++

-ltt

--

I*

+

-

--

ttt ++ + m-- 1-=-=-1-=-

K. YAMAGUCHI and K. FUJINO

74

Anti Dc 2 agglutinin value
Immune rabbit.
I, Blood ( Dilution of antiserum
Existence or /'Absorption) Workof.
. blood I blood , type1 i 1
1
1 I 1 I, 1
Serum Serum I. non-existencecorp, corpuscle I striped
type of Character
usc1e I, dolphin 20 j 40 80 160 ! 320: 640
No.
_ _ _ ,_ _ _ 1. A in saliva
!
I
i
I
)
1
No. 3 1 Dc1 Dc2 ttt J ttt tH ++ i +
No. 6
o'
'
0'
NDc;6 I No. 1 I
Dc1- I j '(!)'

I

I I

1·

1

I

i--

I I

I-

No. 4

'

Dc2

1

ttt

1

Im ++++[+1-=-

Table 4. Haemolysine value of anti Dc1 and Dc2 immune
sera to each type of serum of striped dolphin
Anti Dc 1 haemolysine value
Dilution of antiserum
Immune rabbit[ BI d') Existence or Absorption Work
typ~o of'I
I
I
I
Serum I Serum non-existence c blood 1 co~loos~le I striped1
1 I 1 I 1 ! 1 I 1
pu! dolphin
20 40 80 160132011640
No. ' type of Character, orpusc e
A in s:lliva /I
jI
No. 3 j_D_c_1_D_c_2 ttt \ ttt ttt ! ++ I + I 1'

II

i,:

1

N~ 7 1

(3 1

1

01

I ~~2

8

I

I

1

No. 1 I
No. 4

ttt

Dc1

I Dc

2

-

Im ttltt +1-=1-=-1-=-1-=---=-1-=-

Anti Dc 2 haemolysine value
Dilution of antiserum

Immune rabbit

Blood
Absorption Work
type of
Existence or
blood
blood
Ser.um Serum non-existence corpuscle corpuscle stripedNo.
type of Character
dolphin
A in saliva
1

I

No. 6
'(!)'

I o'
I
I

I

01

I
Paragraph 3.

No. 16
Dc1

1

1

1

-

1~1~1 tH ++ +
-

No. 3

Dc1 Dc2

No. 1

Dc1

I

Dc2

jffi(ttTtt ++ +

No. 4

I

I1

1 160
1 I 8020 40
320 640

-

\
I

-

-

I!

Appearance Rate of Each TypeBy the above stated method it has become clear that the twokinds, that is to say Dc1 and Dc2 , of agglutinogen and haemolysinogen
exist in the blood corpuscles of striped dolphin. The blood corpusclesof the 36 striped dolphins are classified by this into the following threekinds.

75

Serological Constitution of Striped Dolphin

Blood typeMale

Dc1 Dc2
Dc1
Dc 2

10 (50.0)

Total

20

I~
I

4(20.0)6(30.0)I
I

FemaleTotal

11 (68.8)
3 (18. 7)
2 (12.5)

21 (58.3)
7 (19.4)
8 (22. 3)

16

36(Parenthesized figure shows percentage.)

Chapter 6.

Anti Dc1 and Anti Dc2 lsohaemagglutinin Seen
in Normal Sera of Striped Dolphin

The Table 5 shows the respective comparison between the coagulating reaction of each type of the striped dolphin's blood corpuscles to'.table 5. Comparison between the coagulating reaction ofeach type of the striped dolphin's blood corpuscles to the anti
Dc1 and Dc2 immune sera and the coagulating reaction to thenormal sera N os. 4, 16 and 6 of the striped dolphin which
has the isohaemagglutinin.

~~<:~~i:~~Blood corpuscles of striped dolphin

corpuscle

blood
corpuscle

1

I§SI Anti Dc1 l No. 4 (Dc]* Anti Dc1 I No. 16 (Dc

2

3

2)

Dc1 Dc1
Dc2 Dc2

I
I

II

I

Q)

I

I

Dc1
Dc 2

-

I

Z 00 Serum No. 61

7

8

9

10 11

Dc1

Dc2

......
oi S is erum N o. 4'i@ SerumNo.161
0

6

Dc1

Dc1

Blood type!

e5

12 13 14

+tt ++ ++ - ++ ++ - - ++ - ++ ++ - +tt
ttt ttt ++ - ttt +tt +tt - ttt - +tt +tt ttt

2)

I
_)

4

Ht

Dc 2 Dc2

Dc1
Dez
Dc1
Dc1
Dc2
Dc2

Dez

- ++ - ++ +tt --+tt - ttt + +

-

-

-

Blood corpuscles of striped dolphin
15 16 17 18 19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34 35 36++ ++ Ht ttt ++ - ++ ttt tit +tt +tt +tt ++ +tt - tit ++ +tt +tt ttt ++ Ht - ttt ttt ++ ++ - ttt ttt Ht +tt Ht ++ ++ ++ +tt ++ ++ ++ ++
++
Dc1

Dc1

Dc1
Dc 2
Dc1 Dc1 Dc1 Dc1 Dc1
Dc1 Dc1 Dc1 Dc1 Dc1 Dc1 Dc1
Dc2 Dc 2 Dc 2 Dc2 Dc 2
Dc 2 Dc 2 Dc 2 Dc 2 Dc 2 Dc 2 Dc 2

Dc 2

Dc1 Dc1 Dc1
Dc1
Dc 2
Dc 2 Dc 2 Dc 2
- tit -

ttl

- ++ +

+

Dez

- ++
- ++

76

K. y AMAGUCHI and K. FUJINO

the anti Dc1 and Dc2 immune sera and the coagulating reaction to thenormal sera Nos. 4, 16 and 6 of the striped dolphin which has the. isohaemagglutinin.
According to this Table, the Serum No. 4 seems to react positively
to all other Dc1-type blood corpuscles than No. 1 and to have the anti
Dc1 normal agglutinin, but it reacts positively only to No. 13 andnegatively to all others so far as Dc1-Dc2-type is concerned.
While the Serum No. 16 reacts positively only to the Blood corpuscles Nos. 8 and 10 of the Dc2 -type. The Serum No. 6 reacts soweakly that it has no relation to Dc1-Dc2-type at all.
That is to say that some normal isohaemagglutinins in the serumof striped dolphin show the similar reaction to the agglutinin obtainedby immunization, but that their reaction. is so weak and appears so
irregularly that the blood type can not be classified clearly by this.
On the other hand, there exist some other isohaemagglutinins whose
reaction has no connection with DccDc2 blood type.
Chapter 7.

Conclusion

1) So far as just our survey is concerned, the type a' and type ·O' were found, but the type /3' and type a' /3" were not found in theserum of striped dolphin. The existence of anti C agglutinin was not
testified in that serum.
2) The existence of the two kinds (Dc1 and Dc 2) of antigen in the
blood corpuscle of striped dolphin was testified by the anti Dc1 and Dc2
agglutinins and haemolysins obtained by immunizing the rabbit withthe striped dolphin's blood corpuscle. The blood corpuscles of striped
dolphin can be classified by this into the three kinds, namely Dc1-Dc2type, Dcctype and D2 -type.
3) Some isohaemagglutinins of striped dolphin react particularly
to Dcctype or Dc2-type and some others react having no connection
with Dc1-D2-type. It was found, but they appear irregularly.
At last it is a pleasure for the authors to record here a debt ofgratitude to Professor Furuhata of the Institute of Legal Medicine,Tokyo Medico-Dental University, for his kindness in guiding their workand reading the original manuscript, and also to Professors Hiyama andSuehiro of the Fisheries D~partment, Faculty of Agriculture, Tokyo
University, for their kindness in reading the original manuscript.
Further the authors desire to acknowledge the valuable advice, renderedby Professor Ogawa of the Anatomy Department of Faculty of Medicine,Serological Constitution of Striped Dolphin

77

Tokyo University, in naming the Dc1 and Dc2 antigens. They also wish
to thank Dr. Oumura, chief of the First Biological Research Section,
Fisheries Agency, and Dr. Maruyama, chief of the Whales ResearchInstitute, for their usual encouragement.
References
1) Shirai, S.: Keio Igaku, Vol. 3, No. 4, pp. 311-320. (1923).
2) Ottenberg, R. and Friedman, S.: Journal of exp. med., Vol. 1, No. 37, pp. 531535. (1911).
3) Fischbein, M.: Journal of infect. dis., Vol. 12, pp. 133-139. (1913).
4) Snyder, L. H.: Journal of Immunology, Vol. 9, No. 1, pp. 45-48. (1924).
5) Miki, H.: Kyotofuritsu Ikadaigaku Zasshi, Vol. 16, No. 1, pp. 199-232. (1936).
6) Izeki, N.: Juzenkai Zasshi, Vol, 42, No. 12, pp. 3627-3652. (1937).
7) Yamaguchi, K.: Hanzaigaku Zasshi, Vol. 15, No. 4. (1941).
8) Ueyama, R.: ibid., Vol. 12, No. 6. (1938).
9) Ueyama, R.: ibid., Vol. 14, No. 3, pp. 407-425. (1940).
10) Asakawa, K.: Chiba Igakkai Zasshi, Vol. 11, No. 6, pp. 906-1000. (1933).
11) Mizutani, S.: Juzenkai Zasshi, Vol. 37, No. 12 & Vol. 38, No. 2. (1932-33).
12) Mizutani, S.: ibid., Vol. 37, No. 12, p. 2951 & p. 2972. (1932).
13) Ueyama, R.: Hanzaigaku Zasshi, Vol. 13, No. 1, p. 15. (1939).
14) Furuhata, T. : Tokyo Iji Shinshi, No. 3120, p. 271. (1939).
15) Maeda, I.: Juzenkai Zasshi, Vol. 43, No. 4, p. 1333. (1938).
16) Maeda, I. : ibid., p. 1350. (1938).
17) Yoshiwara, M.: Chiba Igakkai Zasshi, Vol. 13, No. 4, p. 945. (1935).