FROM THE STATE SERUM I NSTI TUTE, COPENHAGEN

(CHIEF: J . ORSIZOV, 111. D.)

ON THE SEROLOGY OF THE VIBRIO CHOLERAE
By F. Kauffmann.
(Received for publication December 14th, 1949.)
The studies, of which an account will be given in the following,
were undertaken because Gallut (1) stated that he was able to con-
firm the findings reported by Burrows and collaborators ( 2) , who,
in addition to the cholera types already known - Inaba, Ogawa and
Hikojima - set up a number of new serological types or subtypes.
The demonstration of several types within a single cholera epidemic
- as, for instance, the one that occurred in Egypt in 1947 - raised
doubt about the reliability of the results obtained. But, before entering
into the possibility of these numerous new types, whose antigens are
designated by the letters A-M, it is essential to ascertain the mutual
relationships between the Inaba, Ogawa and Hikojima cultures.
From the available literature no clear-cut idea can be gained about
these relationships, because it is not clear whether we are dealing with
constant, independent types or with unstable variants of one and the
same type.
KabCshima ( 3) , who, according to the current nomenclature, was
working with Inaba and Ogawa strains, established transmutations
from one type to another, when he grew strains in homologous O-im-
mune serum or in rabbit gall bladder. On the other hand, he was
unable to produce any transmutation when the strains were grown on
culture media.
I n 1923 Nobechi ( 4 ) found a third type, which he designated as
the >middle< type (= Hikojima) ; and he gave the following antigenic
formulae for these three types:
Original (= I naba) = A X
Varied (= Ogawh) = B ( X)
Middle (= Hikojima) = A (B) X
284
He confirmed the constancy of these types when grown on arti-
ficial media. Like KabCshima, however, he was able by cultivation in
homologous immune serum to transmute two )>varied<( strains to the
>>middle<( type. I n the course of his experiments he found all sorts
of transitions in the quantitative development of the antigens, and he
designated these transitional forms as follows: ABX - (A)BX -
From these investigations Nobechi arrived at the following con-
clusion: >>Considering the transmutability, the types under discus-
sion do not correspond to the permanent types of dysentery bacilli,
of those of the typhoid - paratyphoid group in a biological sense.
The types of V. cholerae concerned are merely imm'unological modifi-
cations, and do not differ from each other in any of the morphological,
cultural as well as biological aspectscc. He also states: )>As the middle
type is, as mentioned above, situated between the original and the
varied type, the author supposed it to be a type in the passage state
during the transmutation between the two types, happening perhaps
in the gall bladder of a human carriercc.
Aoki & Oshiro ( 5 ) also subscribed to this view, considering these
three types as unstable variants, but they attributed the difference
between these three variants not only to thermostable but also to
thermolabile antigens.
Gardner & Venkatraman (6) established that Inaba, Ogawa and
Hikojima belonged to their 0 group I, and in 1935 they said about
the Hikojima type: )>The table does not include absorption tests of
middle type serum, since our results have been conflicting and the
character of this )>type<<is at present uncertain ... Further work on
this point is desirable<<.
I n 1933, deviating from his previous report, Nobechi ( 7) gave the
formulae for the three types as follows:
(A) (B)X - A(B)X - AB(X).
Inaba =A X
Hikojima = ABX
Ogawa =B CX
Thus a new antigen, C, was introduced in the formula for the
Ogawa type, making the problem even more complicated.
I n contrast hereto, Bruce White (8) confirmed the formulae pre-
viously given by Nobechi, namely Inaba = AX, Hikojima = A BX
and Ogawa = BX.
Burrows and collaborators as well as Gallut were likewise able to
confirm these findings, but they altered the antigenic designations,
designating X as A and A as C. Their formulae, which are quite in
keeping with those given by Bruce White, thus are: Inaba = A C, Hiko-
jima = A B C, and Ogawa = A B. Although the designations suggested
by Nobechi and by Bruce White thus have the priority, and there is no
285
urgent reason to alter them, the nomenclature employed by Burrows
and collaborators and by Gallut has been used in this study. This makes
it easier to compare our results with those obtained by Gallut, and
further, these designations have already gained foothold in the various
periodicals as well as in the textbooks.
Deviating from all the above-mentioned statements, Scholtens (9)
and Heiberg ( 10) established two serologically different cholera types
which they designated as A and AB. The relationship between these
types and the Inaba, Ogawa and Hikojima strains has hitherto been
unsettled.
I n 1947 Shrivastava & Bruce White ( 11) reported that they had
been able by cultivation in >>Ogawa mono-specific antiserumcc to trans-
mute 10 cholera strains and 3 El Tor strains of the Ogawa type into
the Inaba type. Of 8 cholera strains which probably belonged to the
Hikojima type, 4 cultures were transmuted into the Ogawa type by
growing them in >>mono-specific Inaba antiserumcc. On the other hand,
they had been unable to transmute 8 strains of the Inaba type to other
types by growing them in homologous mono-specific serum.
Writer's Studies.
Above all, I wish to give my best thanks to Dr. Gallut, Institut
Pasteur, Paris, for his kindness in placing at my disposal a consider-
able number of his cholera cultures and sera, including also absorbed
sera.
41 cultures received from Dr. Gallut were examined biochemically
and serologically. According to my findings, 36 of these strains, in-
cluding 23 strains of the Inaba type and 11 strains of the Ogawa-
Hikojima type, belonged to Gardner and Venkatraman's 0 group I.
Two strains which were somewhat rough, could not be classified with
certainty. The remaining 5 strains - Hanoi' XII, Ez 5 cp., Ismai'lia 12,
Ismai'lia 13 and 10420, were not cholera strains, and will be discussed
in detail later.
The following 23 cultures are Inaba strains:
V 2 Farana
Port Said 289
95656 .
N. I. H. 35
Port Said 296
10440
El Tor B 1
Port Said 163
8990 Port Said 291
El Tor 4
El Tor 25 Port Said 285
N. I. H. 35 b
N. I. H. 35 a 3
N. I. H. 35 k
Suez V 4 (ACDIKL)
Tantah
El Tor B 2
12976
Suez V 4 (ACDEL)
B 4
V 1 Korein
286
The following 11 cultures are Ogawa-Hikojima strains :
Ogawa
N. I. H. 41
N. I. H. 41 b
Shanghai 10
N. I. H. 389
B 3
Hikojima
Makassar 676
Makassar 794
Sa'igon 1020
Saigon 1046
Inde 653
Vb 610
Strains V 3 Suez and 8946 were somewhat rough and could not
be classified with certainty; but strain 8946 probably belongs to the
Inaba type.
I n addition, 6 cholera strains from Dr. B. Heiberg's collection were
examined, including Inaba strain 289 and Hikojima strain 94. These
6 strains were furnished me by Dr. M. Kristensen.
Altogether 47 cultures were examined, and 28 of these were em-
ployed for the production of immune sera. The majority of the sera
were 0 sera produced after the method recently given by Roschka ( 12) .
As this method has proved to give excellent results in the produc-
tion of Salmonella and Escherichia sera, it was employed in these,
experiments, too.
A 20-hour agar culture was suspended in saline and boiled for
234 hours in flowing steam. After centrifuging, the sediment was in-
cubated with 96 % alcohol at 3 7 O for 4 hours, then centrifuged again,
and washed twice with acetone. Then the sediment was collected
together with a few ml. acetone and kept overnight at 37O. Of the
powder obtained in this way, a small portion was ground in mortar,
stirred with saline and then injected intravenously into the rabbits
i n increasing doses. 4 injections were given at intervals of 4 5 days.
Ten days after the last injection the animals were bled totally.
This method corresponds almost completely to the one given by
Bruce White ( 17) , as for immunization of rabbits he used >>dried,
alcohol-extracted, steam-treated, ether-washed powder<<.
For the sake of control, in addition, several sera were produced
with formalin-treated agar cultures. The sera produced after the
Roschka method contained no H agglutinins, but their 0 titer was
no stronger than the one obtained with sera produced with formalin-
treated cultures. The latter, however, contained also H agglutinins.
The 0 titer of all the sera varied between 1: 640 and 1: 2560,
averaging 1: 1280. The same applies to the sera furnished me by Dr.
Gallut, so that I did not obtain the titers of 1: 10000-1: 20000 as
given by Gallut in his publication. Presumably the cultures employed
by me possessed better developed 0 antigens, and this may explain
the low 0 titer. The stronger the antigen development, the more anti-
gen will be bound per unit of volume, and the lower the agglutination
titer will turn out. The poorer the development of 0 antigen, the
287
higher is the agglutination titer, which is not always a pure 0 titer
but dependent upon the activity of R or Q agglutinins.
Unfortunately, it still happens often that the quantitative aspects
in the antigenic analysis are overestimated, so that the decisive qua-
litative differences are not taken sufficiently into consideration. Thus,
for instance, an 0 serum with an 0 titer of 1: 640 that is perfectly
free from H agglutinins and contains but small amounts of R or Q
agglutinins is better than a serum with a titer of 1: 20000 that does
not react in the same specific manner. Of course, the cultures that
are employed for the agglutination are of great significance, too. Their
colonies must be perfectly smooth and well-developed; if not, even
with only a slight tendency to spontaneous agglutinability, some un-
specific reactions may appear that may give very high titers.
For the agglutination test, a living agar culture was used for the
slide test, a formalinized saline suspension for the tube test. These
suspensions in 1 % formalin saline were made so dense that for
the agglutination test they had to be diluted 1: 100 or more. The tube
agglutination was read after standing in water-bath at 50 for 20
hours.
The absorption experiments were carried out with suspensions
from agar plate cultures that were heated to looo for 1 hour or killed
with 1 % formalin. I n all the experiments the absorbed sera were
tested both with living agar culture and with formalinized culture,
and the two methods always showed concordant results as to the 0
agglutination.
The biochemical tests were carried out after the schema employed
in the Salmonella diagnosis - and in addition to the carbohydrates
and alcohols usually employed, mannose was used.
The hemolytic capacity of the strains was tested by Heibergs
method - i . e., with 5 % sheep erythrocytes, washed three times in
saline. To 0.2 ml. erythrocyte suspension 0.2 ml. of a 20-hour broth
culture was added: then incubation for 2 hours at 3 7 O and additional
standing in ice box at + 50 for 24-48 hours.
Antigenic Structure of the Znaba, Ogawa and Hikojima Strains,
On the basis of investigations reported by Nobechi and other J a-
panese authors and confirmed by Bruce White, the antigenic rela-
tionships between the Inaba, Ogawa and Hikojima strains may he ex-
pressed as follows:
Inaba = A C (= A X )
Ogawa = A B ( = B X )
Hikojima = A B C (= A B X )
288
Agglutinated
with culture
Inaba
Ogawa
Hikojima
The present studies, the more essential results of which are re-
corded in Table 1, give conflicting results - similar to those obtained
by Gardner & Venkatraman. Still, the latter authors refrained from
publishing their absorption results with a Hikojima serum.
My own conflicting results are that the Hikojima sera are regularly
completely absorbed by Ogawa strains so that the C agglutinin, which
really should be left in the serum is lacking. This striking fact, which
was established repeatedly as a constant phenomenon in experiments
with several sera, may be explained in the following way:
Most likely, the Hikojima strains contain only a small amount of
C antigen, as in pure C serum (I naba serum absorbed by an Ogawa
strain) they react considerably more weakly than do the Inaba cul-
tures. I t is easy to understand, therefore, that the sera produced with
Hikojima strains contain but a scanty amount of C agglutinin. Further-
more, probably the Ogawa strains contain also some C antigen, making
them able completely to absorb a Hikojima serum. On the other hand,
the small amount of C antigen is not sufficient completely to empty
an Inaba serum, whereas the Hikojima strains, which contain more
C antigen, are able on massive absorption to empty such a serum
completely.
If Ogawa strains are grown at 20, however, it is possible with
such cultures to absorb an Inaba serum completely or almost com-
pletely (residual agglutinin 1: 10). At this temperature of 20 the
B antigen of the Ogawa strains develops more poorly so that, probably
for this reason, the development or >>disponibility<< of the C antigen
is not inhibited by the B antigen.
0 Sera
Inaba abs. with Ogawa abs. with Hikojima abs. with
Ogawa I Hikojima Inaba I Hikojima -4 Inaba I Ogawa
- - - - -
-
-
++
++
-
-
++
++
-
++
+
-
-
289
there is nothing left but to give up a differentiation between Ogawa
and Hikojima strains, i. e., to distinguish only between two types,
namely : Inaba and Ogawa-Hikojima (or, briefly, Ogawa) . Then the
antigenic formulae of these two types might be given as follows:
Inaba = A C
Ogawa-Hikojima = A B ( C)
The parenthesized C antigen indicates that this antigen is not al-
ways fully developed or >>disponible<, so that Ogawa strains grown at
37O -in contrast to those grown at 20 - are unable completely to
empty an Inaba serum.
I t is to be emphasized that Ogawa and Hikojima strains in cross-
absorption experiments prove identical, as is evident from Table 1.
I t is justified, therefore, to group the Ogawa and the Hikojima strains
together as one type.
For the differential diagnosis between Inaba strains and Ogawa-
Hikojima strains only one serum is employed, namely: a B serum
prepared by absorption of an Ogawa or a Hikojima serum with an
Inaba strain. So the formulae for these two types may be simplified
by designating the Inaba type with A, the Ogawa-Hikojima type with
AB. This bridges the gap between the findings reported by Nobechi
and by Bruce White, on the one side, and the findings obtained by
Scholtens and by Heiberg, on the other side, so as to leave no conflict
between the two views advanced by these authors.
As mentioned above, Scholtens and Heiberg divided the cholera
strains of Gardner & Venkatramans 0 group I into two subgroups,
namely: 1) strains containing antigen A, and 2) strains containing
antigens AB.
The present studies have established that Heibergs strain (No.
289) of group A is an Inaba strain, while a strain (No. 94) of group
A B is a Hikojima strain. The identity of these two strains with the
Inaba and the Hikojima types, respectively, was demonstrated by
cross-absorption experiments. So, on comparison between the two
types - Inaba and Hikojima - it is evident that the formulae of A
and A B are correct and fully adequate for a thorough interpretation
of all the absorption results obtained.
As mentioned already above, these matters become complicated
first when the Ogawa strains are included in the picture, because
these strains grown at 3 7 O empty a Hikojima serum, it is true, but
not an Inaba serum. I n order to avoid these difficulties, in practice
only the simplified formulae - A for Inaba and A B for Ogawa-Hiko-
jima- are taken into consideration. These formulae are the more
justifiable because the existence of a special C antigen has not been
proved decisively.
We may assume that the A antigen develops more poorly -or is
Acta path. XXVII. 2 . 19
290
less readily )>disponibel<( - in the Ogawa strains when the B antigen
is strongly developed, so that the formula for the Ogawa type might
also be given as ( A) B. So, by designating the Inaba strains as A, the
Hikojima strains as A B and the Ogawa strains as ( A) B, we obtain
exactly the same results as by giving the formulae as AC, ARC and
AB( C) .
As the chief 0 antigen in 0 group I is antigen A, while the antigens
B and C are merely less developed partial antigens, this question of
formulae is only of minor importance. I n practice, at any rate, we
should limit ourselves to the formulae of A and AB set up by Scholtens
and by Heiberg.
I t must have been attributable to unfortunate circumstances that
Heiberg overlooked that his A B strain was identical with Ogawa-Hiko-
jima, as the only one of his strains designated as Ogawa cannot have
been a true Ogawa strain. According to the statements of Heiberg,
this strain is not agglutinated by A and A B sera, and thus it did
not belong to Gardner and Venkatramans 0 group I, but to some
other infrequent 0 group. As this strain, which erroneously was
looked upon as an Ogawa strain, was not kept in storage, it could
not be reexamined. I n the intervening period the Hikojima strain ex-
amined by Heiberg (290) had become perfectly rough, so that it, too,
could no longer be reexamined serologically. From the absorption re-
sults reported by Heiberg, it is not evident whether this strain was
really a Hikojima strain.
The fact that a Hikojima serum ( 290) was apparently absorbed
completely by an unquestionable Inaba strain may be explained in
this way: the B titer of this Hikojima serum was lower than 1: 100,
for Heiberg performed his agglutinative tests at the lowest dilution
of 1 : 100. We have to reckon with this possibility as the titer of the
unabsorbed serum was only 1 : 400-1 : 800. Nevertheless, the pos-
sibility still remains that the Hikojima strain 290 really was an Inaba
strain, so that Heiberg rightly designated its formula as A.
Today we can merely establish with certainty that the Inaba strain
( = original) examined by Heiberg really is an Inaba strain, and that
strain 94 designated by him as A B is a Hikojima strain.
So the formulae A and A B employed by Scholtens and by Heiberg
are correct and not in conflict wi t h the formulae given by Nobechi
and Bruce Whi t e. I n practice, however, they are preferable, being more
simplified.
Sub-Types Set u p by Burrows and Collaborators
and Gallut.
As mentioned in the introduction, I am greatly. indebted to Dr.
Gallut for sending me 41 cultures and some sera, both absorbed and
unabsorbed, thus enabling me to work with Dr. Galluts own material.
As Gallut arrived at the same results as reported by Burrows and
collaborators and also examined in part the same strains as these
authors, my examination of Galluts tests applies also to those re-
ported by Burrows and collaborators.
According to the present test, 36 of the 41 cultures examined belong
to Gardner and Venkatramans 0 group I. Of these 36 strains, 23
belong to the Inaba type, while 11 belong to the Ogawa-Hikojima
type, and 2 are somewhat rough so that they could not be typed with
certainty. With the exception of 2 strains, this classification into Inaba
and Ogawa-Hikojima strains corresponds to the one reported by Gal-
lut. The two exceptions are strains El Tor 4 and El Tor 25, which
in my experiments turned out to be Inaba strains, whereas Gallut
diagnosed them as Hikojima strains. After I had informed Gallut
about these deviations, he sent me once more strain El Tor 4, but in
my experiments this culture, too, turned out to be an Inaba. The re-
maining 5 strains, which do not belong to the cholera 0 group I, are
as follows:
1. Strain Hanoi XII, belonging biochemically to Heibergs type I,
behaves like a cholera strain but is hemolytic, and -in contrast to
cholera cultures - it gives a weakly positive Voges-Proskauer reac-
tion. According to Galluts paper of 1948, this strain has the formula
BCDL. But in a private communication of 1949 Gallut finds this strain
to have the formula A, even though he fails to disclaim the former
formula.
I was unable, however, to confirm either of the two formulae, as
this strain neither belongs to Gardner and Venkatramans 0 group I
nor is it in possession of antigens BCDL. Serologically, thus, this strain
has no antigenic relationships to the Inaba, Ogawa or Hikojima strains,
as these cultures were not agglutinated by a Hanoi XI1 serum. Con-
versely, strain Hanoi XI1 is not agglutinated by Inaba, Ogawa or Hiko-
jima sera, so that the formulae BCDL or A given by Gallut could not
be confirmed.
2. Strain Ez 5 cp, the formula of which is given by Gallut as GHJM,
belongs biochemically to Heibergs type 11, as it does not split man-
nose. It is hemolytic, but it is not an El Tor strain since - in con-
cordance with Gallut -it does not belong to Gardner and Venkatra-
mans 0 group I. I n contrast to the statement made by Gallut, n o
antigenic relationships could be demonstrated between this strain and
Inaba, Ogawa and Hikojima strains. The immune serum produced
with strain Ez 5 cp did not agglutinate any of the cholera strains;
nor did this strain react with any of the cholera sera.
3. Strain 10420, which Gallut designated as atypical, even though
he claimed it to contain antigen A, is not a cholera strain biochemical-
ly as it does not split the carbohydrates or alcohols tested. Serologic-
19
292
ally, no relationship could be demonstrated between this strain and
the cholera group I, and thus I was unable to confirm the presence
of antigen A. At my request, Gallut was kind enough to send me
another culture of this strain, and it gave exactly the same result as
the one first received. The immune serum produced with this strain
agglutinated none of the cholera cultures; nor was the strain itself
agglutinated by any of the cholera sera.
4. Strain Ismailia 13, which is claimed by Gallut to contain the
B antigen, proved to be an Escherichia coli strain and possessed no
cholera B antigen. I informed Dr. Gallut about these striking findings,
but he was unable to send me any other pure culture of the strain.
5. Strain Ismailia 12 -which, according to Gallut, should con-
tain antigens CGJ - proved to be an atypical Escherichia strain which
had no 0 antigen in common with cholera cultures. After I had in-
formed Dr. Gallut about these deviating biochemical characters, I re-
ceived from him another culture of this strain which gave the same
results. As Gallut employed this strain -1smai'lia 12 (CGJ ) - for
absorption of an Inaba serum, in order to obtain the A agglutinin in
pure form, it will be obvious from the above that it was impossible
for me to prepare agglutinin A in pure form. So far, it has not been
practicable to produce A agglutinins i n pure form because strains
containing antigen C, or antigen B, and at the same time lacking the
A antigen are not available.
I have not been able by means of factor sera furnished me by
Dr. Gallut to confirm the existence of new subtypes. I have therefore
produced myself a fairly large number of sera, which I have absorbed
i n various ways in order to obtain the factor sera given by Gallut.
The outcome of these experiments may be summarized briefly as
follows: I n no case whatever have I been able to demonstrate factors
D, E, F, G, H, I, J , K, L, and M. I succeeded merely in demonstrating
the presence of antigens already confirmed repeatedly - namely, A,
B and C (see above) - i. e., to divide the 0 group I into the Inaba
and Ogawa-Hikojima types (for details, see the preceding section).
For the production of factor sera, the sera showing just as high
a titer as those produced by Gallut were absorbed in dilution 1: 5 or
1 : 10. The absorbed sera were tested both by slide agglutination ( 1 : 5
or 1: 10) with living agar culture and by tube agglutination with
formalin-treated cultures (from agar plates) in dilutions 1 : 10 or 1 : 20,
to 1: 5120.
With the exception of factor sera B and C, all the absorption ex-
periments turned out perfectly negative, as the sera were emptied
completely; they agglutinated neither the strain used for the production
of the sera nor the strain employed for the absorption. All the strains
designated by me as Inaba were perfectly identical serologically; and
also all the Ogawa or Hikojima strains were mutually identical. There
293
was no evidence whatever of the possible occurrence of types other
than those just mentioned : Inaba and Ogawa-Hikojima.
I n order to illustrate the identity of the Inaba or Ogawa-Hikojima
strains examined, some of the absorption experiments are recorded
in Tables 2 and 3. At the same time, these tables further show that
I have been unable to produce the factor sera D, E, F, etc., given by
Burrows and by Gallut. The absorbed sera which on tube agglutina-
tion were tested in dilutions from 1 : 10 or 1 : 20, and on slide agglutina-
tion in dilutions from 1 : 5 or 1 : 10 failed to show even the least trace
of agglutination when the reactions were read by means of a lense.
Positive reactions appeared only in absorption experiments when
the tests recorded in Table 1 were performed, i. e., by production of
a B serum, or by absorption of an Inaba serum with an Ogawa strain.
So no definite evidence could be found to the effect that the two
types set up - the Znaba type and the Ogawa-Hikojima type - might
be further divided into subtypes.
As to the cholera epidemic in Egypt in 1947, moreover, it may
be said that 17 strains isolated from this epidemic were Inaba strains,
and they all proved to be perfectly identical. According to Gallut, these
17 identical strains should belong to 12 different serotypes, which
epidemiologically may be said to be a highly improbable finding. In-
deed, it has not been confirmed by the present experimental studies.
Of these Egyptian strains, 8 were used for the production of im-
mune sera, and it was demonstrated by cross-absorption that these
strains are identical. From these experiments it is evident that the
cholera epidemic in Egypt in 1947 was caused by the Inaba type, and
that - in contrast to the statements made by Gallut - these Inaba
strains could not be divided further into subtypes.
Table 2.
Results of 0 absorptions (Inaba)
Scruni
Formula
Gallut
V 2 Farana
95656
N. I. H. 35
N. I. H. 35 b
N. I. H. 35 a 3
N. I. H. 35 k
El Tor 4
Suez V 4
Port Said 291
12976
B 4
V 1 Korein
A C
A C
A C
A C D
A C D E
A C D F I K
A B C F L
A C D I K L
A C L
A C I K
A C E
A C D L
Absorbed
with strain
Formula
Gallut
A C
A C
A C
A C
A C
A C
A C
A C
A C
A C
A C
A C
Results of
agglutination
294
I
Serum
~~
Formula
Gallut
Serum
N. I. H. 41 A B E
N.I.H. 41 b A B E L
N.I .H. 389 A B E F H
Formula
Galluf
Absorbed Formula Results of
with strain Gallut I agglutination
Makassar 676 A B C E -
A B C E -
A B C E -
>)
))
Makassar794
A C
A C
A C
A C
A C
A C
A C
A C
A C
A C
A C
A C
)>
-
A B C E L I A B C E
Absorbed,
with slrain
A B E
A B E L
A B E F H
A B C E L
V 2 Farana
95656
N. I. H. 35
N. I. H. 35 b
N. I. H. 35 a 3
N. I. H. 35 k
El Tor 4
Suez V 4
Port Said 291
12976
B 4
V 1 Korein
-
-
-
-
Formula
Gallut
A C
A C
A C
A C D
A C D E
A C D F I K
A B C F L
A C D I K L
A C L
A C I K
A C E
A C D L
Key: - - - no agglutination of the strains used for immunization and ab-
sorption.
Table 3.
Results of 0 absorptions (Ogawa-Hikojima).
Makassar 676
))
))
)>
N. I. H. 41
A B C E N. I. H. 41 b
A B C E N.I.H. 389
A B C E A B C E I Makassar 794
Key: - - - no agglutination of the strains used for immunization and ab-
sorption.
Biochemical and Serological Diagnosis of Cholera Vibrios.
All the cultures here examined, which belong to Gardner and Ven-
katramans 0 group I, correspond biochemically to Heibergs type I
(13) : they split mannose and sucrose, but not arabinose. Their bio-
chemical and serological characters are as follows :
No fermentation of adonitol, arabinose, dulcitol, inositol, rhamnose,
salicin, sorbitol and xylose. Rapid fermentation of glucose (without
gas), maltose, mannitol (without gas), mannose and sucrose. Delayed
fermentation of lactose after 2-44 days. Production of indole and
liquefaction of gelatin. No production of H,S and no decomposition
295
of urea. Irregular behaviour on Simmons agar with glucose and so-
dium citrate. Mostly positive nitrate reaction, but negative Voges-
Proskauer and methyl-red reaction.
Of the cultures examined only 6 strains were capable of hemolysis,
namely: El Tor B 1, El Tor B 2, El Tor 4, Makassar 676, Makassar
794 and Heibergs strain 94. Of these 6 strains, the first three belonged
to the Inaba type, the others to the Hikojima type.
I n the performance of the practical cholera diagnosis it is recom-
mended strongly to carry out the above-mentioned biochemical reac-
tions in order to avoid diagnostic errors. I n dealing with non-hemolytic
cultures that are in concordance with Heibergs type I and the other
above-mentioned characters, there can be no doubt about the strains
being cholera cultures if also the slide agglutination test in an 0 serum
of Gardner and Venkatramans 0 group I turns out positive. If
such an 0 serum shows a titer of 1 : 640 - 1 : 2560, it may be employed
for the slide agglutination test in dilution 1: 10 or 1 : 20.
Titration by the tube agglutination method is not necessary, but
for the sake of certainty it may be performed with thestrains first
isolated in an epidemic or in dubious cases. For the tube agglutination
it is best to use a formalinized suspension of an agar plate culture,
whereas a living agar culture is employed for the slide agglutination.
The agar plates should be soft (about 1.7-2.0 % agar, depending on
the agar preparation), thick and not too dry, with a pH value of
Also the B antigen is determined by the slide agglutination method
by means of a B serum obtained through absorption of an Ogawa
serum with an Inaba strain in dilution 1 : 5-1: 10.
On employment of culture media suitable for the diagnosis of
cholera, thorough biochemical tests and serviceable 0 sera, the dia-
gnosis cannot give any particular difficulty. If we have determined
a strain as belonging to Gardner and Venkatramans 0 group I it is
possible by means of a B serum to rapidly and accurately make the
differential diagnosis between the Inaha and the Ogawa-Hikojima
types. The occurrence of new subtypes in the sense advocated by Bur-
rows and by Gallnt could not be confi rmed and hence they are not
taken into consideration in the diagnosis of cholera.
6.8-7.0.
Discussion.
Cholera cultures belonging to Gardner and Venkatramans 0 group
I should not be divided into t ypes, but only into t wo di fferent 0 f or ms
of one t ype: Inaba = A C and Ogawa-Hikojima = A B (C).
Here, for the first time, the term ))O form(<is employed for these
>>types(< or ))variants<<, as -according to the statements in the lite-
rature, in particular the investigations reported by Shrivastara & Bruce
296
White ( 11) and also the present studies - it is not a matter of
constant types but merely of different 0 forms. Owing to the B antigen
there are qualitative differences between the Inaba strains (briefly =
A), on one side, and the Ogawa-Hikojima strains (briefly = A B) on
the other side. The conditions here, then, present a parallelism with
the forms IV, V and IV described by Kauffmann (14) in Salmonella
paratyphi B or Salmonella typhi murium. Under practical conditions
the IV, V forms within the Salmonella group are very constant, and
yet these forms may give rise to a loss variant = IV, which in turn
is perfectly constant. The same would apply to the A B and A forms
within the cholera vibrios, so that these forms have epidemiological
significance, although the A B form is not perfectly stable.
While, on account of the B antigen, there are quaZifatiue differences
between the Inaba strains, on one side, and the Ogawa-Hikojima
strains, on the other side, the Ogawa and the Hikojima strains may
be interpreted merely as variants of one and the same form (Ogawa),
differing mutually by quantitatively differing development of the C
antigen. This antigen thus plays a similar r61e as does the XII, antigen
in the Salmonella group ( 15) , and is subject to >>form variation<. In
order to obtain constant results in practice, only two forms have to
be diagnosed: Inaba = A, and Ogawa = A B.
These shortened formulae are fully adequate for practical require-
ments, and they correspond to the formulae given by Scholtens and
by Heiberg, and thus the results obtained by these investigators are
confirmed.
Through cross-absorption experiments, Heibergs strain 289 = A
was demonstrated to be an Inaba strain, and his strain 94 = AB be-
longs to the Ogawa form, or -more exactly- it is a Hikojima
strain. Thus there is no difference between the findings reported by
Nobechi and Bruce White, on one side, and the results obtained by
Scholtens and by Heiberg, on the other side, both forms of expres-
sion being correct. I n practice, however, the simplified formulae of
Scholtens and of Heiberg are preferable.
While thus I have been able to confirm the findings reported by
Nobechi, Bruce White, and Gardner & Venkatraman, I have not been
able to confirm the more far-reaching statements made by Burrows
and collaborators and by Gallut. The occurrence of new types or
variants within the 0 group I that were claimed to be characterized
by the antigens D, E, F, G, H, I, J , K, L, and M could not be de-
monstrated. I n particular, 17 strains that were isolated in the Egyptian
epidemic in 1947 proved to be Inaba cultures that could not be sub-
divided. The occurrence of the antigens mentioned (D, E, F, etc.)
could not be demonstrated by means of the factor sera received from
Gallut, nor with the factor sera I produced myself.
The reason for these striking differences may be attributed to
technical errors in the planning and estimation of the serologic exa-
297
mination. If, for instance, antigenic analyses had been carried out with
cultures grown on rather unsuitable media as hard, thin and too dry
agar plates, so then the decisive 0 antigens will have developed but
insufficiently. Such cultures will then react in all sorts of sera as the
overlapping somatic antigens - which normally are concealed under
optimal development of 0 antigens - in this way are disclosed. Also
sera produced with such cultures may be unsuitable for antigenic ana-
lysis, as they will be characterized by a marked content of unspecific
agglutinins. This will give some most disturbing results, if the serum
is not absorbed with a massive dose of culture sufficient to remove
all such unspecific agglutinins. But even in the absence of unspecific
agglutinins the examiner often committs the error of working with
insufficiently absorbed 0 factor sera.
Furthermore, it is to be pointed out that not all granular agglu-
tinations represent an 0 agglutination, as some of them may be due
to R or Q agglutinins (16). The differentiation between such reactions
and true 0 agglutinations is by no means easy, requiring considerable
training and experience. These unspecific R or Q agglutinins have to be
reckoned with especially within the cholera group, as here the cultures
readily become rough and spontaneously agglutinable; so that con-
tinuous control and considerable caution are urgently required in the
estimation of the serologic reactions. For the serologic cholera diagno-
sis - in confirmation of the findings reported by Gardner & Venkatra-
man, Bruce White and other investigators - it is essential to have
available an Inaba serum or an Ogawa serum. For this purpose, how-
ever, it is preferable to produce a polyvalent 0 serum, i. e., to immunize
the rabbit with both forms simultaneously.
For the differential diagnosis between Inaba and Ogawa, this poly-
valent serum or an Ogawa serum is absorbed with an Inaba strain in
order to obtain the B agglutinin in pure form. I n practice, we should
leave out of consideration a differential diagnosis between Ogawa and
Hikojima.
For the sake of control, all freshly isolated strains should be sub-
mitted to thorough examination of their biochemical characters and
also their capacity for hemolysis. For, the so-called El Tor strains,
which belong to the 0 group I, differ from true cholera vibrios only
in their capacity for hemolysis.
The H antigens may be left out of consideration in the serologic
diagnosis of cholera, so that merely the employment of 0 sera is re-
quired. The H agglutination of cholera vibrios is all too weak, unspe-
cific, and uncharacteristic to be employed in the practical diagnosis.
The H subtypes set up by Burrows and collaborators, have not been
confirmed as yet, and they are to be looked upon with a great deal
of skepticism.
For the prophylaxis against cholera it is sufficient in the vaccine
production to take Inaba and Ogawa strains into consideration and
thus produce a polyvalent vaccine. The 0 subtypes set up by Burrows
and collaborators and by Gallut could not be confirmed, and hence
they are not taken into account in the vaccine production.
The present studies, which are in keeping with the findings of Bruce
White, have led to a considerable simplification of the cholera sero-
logy. It is appropriate, therefore, to conclude the account of the pre-
sent studies with a quotation from Shrivastava & Bruce White:
,It is relatively seldom that the bacterial serologist, so often under
the stigma of bringing complexity to taxonomy hitherto straight-
forward, finds himself, as we at the moment do, in the happy position
of urging a simplification of ideascc.
As the Inaba and Ogawa-Hikojima strains undoubtedly merely in-
volve different 0 forms of one type, I find no reason to hesitate in
subscribing to the conclusion arrived at by Shrivastava & Bruce White:
)>The existing classification of the vibrios of 0 group I into sero-
logical types is taxonomically invalid<<.
Summary.
1. Cholera vibrios belonging to Gardner and Venkatramans 0
group I should not ,be divided into types but into two 0 forms of only
one type. These two 0 forms of vibrio cholerae are Inaba = A C and
Ogawa-Hikojima (briefly, Ogawa) = A B (C).
2. The Ogawa and the Hikojima strains differ only quantitatively,
with regard to the C antigen, so that these strains may be looked upon
as uariants of the same 0 form. I n practice, therefore, only two 0
forms have to be diagnosed: Inaba = A and Ogawa = A B.
3. The antigenic formulae given by Nobechi and by Bruce White are
correct, and so are the formulae given by Scholtens and by Heiberg. I n
practice, however, the shortened formulae A and A B given by Schol-
tens and by Heiberg are preferable.
4. The occurrence of subtypes within 0 group I claimed by Bur-
rows & collaborators and by Gallut and characterized by antigens D,
E, F, G, H, I, J , K, L, and M could not be confirmed.
5. I n conformity with previous statements in the literature the
serologic cholera diagnosis requires the employment of an 0 serum of
0 group I, preferably a polyvalent serum produced by Inaba and Ogawa
strains.
6. The differential diagnosis between Inaba and Ogawa requires
a B serum obtained by absorption of a polyvalent serum or an Ogawa
serum by an Inaba strain.
7. The serologic cholera diagnosis should always be supplemented
by biocliemicd tests and by examination for hemolysis.
299
8. In the preparation of uaccines only Inaba and Ogawa strains
should be considered, so that a polyvalent vaccine prepared with both
0 forms is sufficient. The subtypes set up by Burrows & collaborators
and by Gallut should not be considered.
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