Japan. J. Med. Sci. Biol.

, 19, 209-214, 1966

NOTE

IMMUNITY TO EXPERIMENTAL KERATOCONJUNCTIVITIS

SHIGELLOSA IN GUINEA PIGS

Specific susceptibility of the eyes of guinea pigs and rabbits to dysentery bacilli makes
it possible to use them as an experimental model for the studies of pathogenesis and

immunity of bacillary dysentery (Manolov, 1957; Sereny, 1957; Watkins, 1960; Mackel,
et al., 1961; Stenzel, 1962; Lin et al., 1964 a, b ; LaBrec et al., 1964; Ogawa et al.,

in preparation). As regards the immunity to dysenteric keratoconjunctivitis in guinea

gigs, Manolov (1957) reported that only after several repeated attacks of the disease did
the eye develop definite local immunity, while the other eye remained susceptible, and
that a parenteral immunization with dysentery vaccine did not lead to local immunization

of the eye. Serð¥ny (1958), Watkins (1960), Mackel, et al. (1961), Stenzel (1962) and
Lin et al. (1964 a) reported that recovery from the first attack of keratocon junctivitis

shigellosa imparted non-specific tissue immunity against reinfection with shigellae, while
humoral immunity was type-specific to the infecting bacilli though in too low a degree
to protect the eyes from massive infection.

The present study, with use of keratoconjunctivitis shigellosa in guinea pigs as a

model, was undertaken to compare the protective efficiency and immunological specificity
in the systemic and local immunization with virulent, attenuated and heat-killed Shigella

flexneri and virulent S. sonnei and Escherichia coli.

Experiment I: Effects of Systemic and Local Immunization :

Female guinea pigs, Hartley strain, weighing 350 to 400g were used.
The systemic immunization was given to each group of five guinea pigs by four weekly

intravenous injections of 1•~108 live cells of S. flexneri 2a strain 5503 (virulent), or its

colonial variants, 5503-I (smooth form, attenuated) and 5503-II (rough form, attenuated).
The animals were challenged by a con junctival infection with live cells of strain 5503

suspended in saline one week after the last injection, and observed macroscopically for
the severity of keratocon junctivitis for two weeks. The characteristics of the virulence

of these strains will be described elsewhere (Ogawa et al., in preparation). The strain

5503 has the capacity not only to induce colitis in monkeys when given orally and
keratoconjunctivitis in guinea pigs, but also to penetrate cultured cells and to multiply

intracellularly. The two colonial variants totally lacked these capacities.

In Fig. 1, the degrees of severity of changes at each stage in individual cases after
the challenge are represented by columns ; in Fig. 1A the challenge was made with 1•~

109 cells, and in Fig. lB with 1•~106 cells. The sera of the immunized animals taken

immediately before challenge possessed agglutinin titers against S. flexneri 2a of 1:1,280.

Though the pretreatment with none of the strains could prevent the development of

keratoconjunctivitis when the challenge was made with a higher dose, the pretreatments

209
 210 NOTE Vol. 19

with strains 5503 and 5503-I resulted in milder changes and faster recovery in comparison
with the untreated control group . The animals immunized with strain 5503-II gave an
intermediate response. Infection with a lower dose was completely prevented by the

pretreatment with strain 5503, but not so completely by those with strains 5503-I and
with 5503-TI: the latters failed to protect some of the animals from the infection .
For local immunization, 1•~109 viable cells of strain 5503 were dropped once into the

right eye of five guinea pigs. Similarly 1•~109 viable 5503-I or 1•~109 heat-killed 5503
were applied twice a week for 4 weeks to the right eye of a group of five guinea pigs
.
After the immunization period of 4-5 weeks, both eyes were challenged with strain 5503 .
The serum agglutinin titers remained negative at a dilution of 1 : 20 by this time
. The
left eye serving as control was observed simultaneously .

Fig. 1. The effects of systemic and local immunizations on the conjunctival

infection with S. flexneri 2a strain 5503.

As displayed in the lower part of Fig . 1, the development of inflammation was not
completely inhibited in the right eye recovered from the infection with strain 5503 when
challenged with a higher dose, but clinical symptoms were milder and their duration was
 1966 NOTE 211

clearly shortened than in the case of the systemic immunization. The local application
of strain 5503-I, however, did not result in such a clear increase of resistance, while the
pretreatment with the heat-killed 5503 showed no effect at all. When a lower dose was
used for challenge, the pretreatments with viable 5503 and 5503-I prevented reinfection.
The local application of the heat-killed cells did not show definite effect ; the fact that
only fewer animals developed clinical symptoms in this case as compared with the control
group may be accounted for by the effect of the systemic immunization which might be
brought about by the local treatment, since a similar number of response was observed
in both eyes. This phenomenon was also shown in the control eye of the groups locally
treated with 5503 and challenged with a higher dose and those treated with 5503-I and
challenged with a lower dose.

Experiment II: Immunological Specificity of the Acquired Resistance :

With the purpose of elucidating the immunological specificity of the acquired resistance

to reinfection which developed after each treatment in Experiment I, reciprocal cross-

protection tests were carried out among S. flexneri 2a, S. sonnei and E. coli. Strain
264-65 of S. sonnei and strain 905-65 enteropathogenic E. coli 0143 employed in these

experiments are pathogenic to the eyes of guinea pigs, and capable of intracellular

multiplication in cell culture. No antigenic relationship was found among these strains

by agglutination tests. The immunization procedures were the same as in Experiment

I. The challenge dose used was 1•~109 per eye.

Fig. 2. Immunological specificity of the acquired resistance to the conjunctival

infection as studied by reciprocal cross-protection tests.
 212 NOTE Vol. 19

In intravenous immunization (Fig. 2A), animals pretreated with viable S. iexneri 2a

strain 5503 showed no acquired resistance to conjunctival infection with the heterologous
bacilli, whereas those pretreated with viable S. sonnei or E. coli showed an increase of
resistance to the homologous infection as had been expected from the results obtained in
experiment I. The serum agglutinin titers against the homologous bacilli ranged from
1:1,280 to 1:2,560 before the challenge. It was also noted that the changes caused by
the challenge with strain 5503 were less severe and their recovery was slightly more
rapid in the latter two groups than in the control group .
In the local immunization, the comparison between the right eye which was pretreated
and the left eye used as control indicated an obvious increase of resistance both to the
homologous and to the heterologous reinfection (Fig. 2B) . The acquired resistance fol-
lowing the recovery from the first attack of keratoconjunctivitis remained unvaried until
9-10 weeks later, whether the reinfection was made with the homologous or heterologous
organisms ; it declined after 15 weeks though still present.
In conclusion, by means of keratoconjunctivitis shigellosa in guinea pigs, comparative
studies were made on the effects of the virulent strain, its attenuated colonial variants
and heat-killed cells of the virulent strain in the systemic and local immunizations . The
systemic pretreatment resulted in development of an immunologically specific resistance,
which was highest when the viable virulent bacilli were employed ; the attenuated vari-
ants, particularly the rough form gave the lowest effect. The con junctival infection of
the viable virulent bacilli induced the resistance higher than that induced by the intraven-
ous treatment : it is clearly evidenced that non-specific factors were involved in this case
of reciprocal cross-protection tests employing three virulent strains of antigenically distinct
dysentery bacilli and E. coli. The local application of the attenuated variant induced
a moderate degree of resistance, whereas that of the heat-killed virulent cells was not
effective at all. No correlation was found between the serum agglutinin titers and the
protective powers in the eyes.

Figs. 3-6: The grades of severity of changes in the infected eyes .

Fig. 3 : Intravenous immunization with S. flexneri 2a, strain 5503 .

a: 48 hr after challenge with 5503; grade + .
b: 48 hr after challenge with S. sonnei ; grade +++.c
: 48 hr after challenge with E. coli ; grade+++.

Figs. 4, 5 and 6 : Local immunization with S. flexneri 2a, strain 5503 in the right eye .

Fig. 4a: A treated eye on 3 days after challenge with 5503; grade•}
4b: The opposite control eye ; grade++ .

Fig. 5a: A treated eye on 3 days after challenge with S . sonnei ; grade +.
5b: The opposite control eye ; grade+++.

Fig. 6a: A treated eye on 3 days after challenge with E . coil ; grade +.
6b: The opposite control eye ; grade+++ .
 1966 NOTE 213

Fig. 3a Fig. 3b Fig. 3c

Fig. 4a . 4h

Fig . 5a Fig. 5b

Fig. 6a Fig. 6b

Fig
214 NOTE Vol. 1

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LABREC, E. H., SCHNEIDER, H., MAGNANI, T. J. AND FORMAL, S. B. (1964) : Epithelial cell

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1503-1518.LIN

, F. C., YANG, T. L. AND YEH, S. H. (1964a): Experimental keratoconjunctivitis shi.gel-

losis in guinea pigs. I. Immune phenomenon and antibody formation. Chinese Med. J., 83,

454-462.LIN

, F. C., YANG, T. L. AND WU, P. F. (1964b) : Experimental keratoconjunctivitis shigel-

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MANOLOV, D. C. (1957) : A study of some questions of pathogenicity and immunity in dysentery

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Microbiol. Immunol. 1, 322-328. (Cited from Biol. Abstr. 33, 857 (10685), 1959)

SERð†NY, B. (1957) : Experimental keratoconjunctivitis shigellosa. Acta Microbiol. Hung., 4,

367-376.

SERð†NY, B. (1958) : Acquired natural immunity following recovery from keratoconjunctivitis

shigellosa. Acta Microbiol. Hung., 5, 343-358.

STENZEL, W. (1962) : Die Schleimhautpathogenitð t der bakteriellen Ruhrerreger im Tierversuch.

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WATKINS, H. M. S. (1960) : Some attributes of virulence in Shigella. Ann. N. Y. Acad. Sc.,

88, 1167-1186.

Department of Pathology HIDEMASA OGAWA

Department of Bacteriology I AKIKO NAKAMURA
National Institute of Health, Tokyo RINTARO NAKAYA

Received : June 21st, 1966

小 河 秀 正(国 立 予 防 衛 生 研 究 所 病 理 部)
中村 明子 ・中谷 林 太 郎(国 立 予 防 衛 生 研 究 所 細 菌 第 一 部)