Veterinary Microbiology, 14 (1987) 81-86 81 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

Short Communication

Opsonic Activity of Anti-flagellar Serum against Clos tr id ium chauvoei by Mouse Polymorphonuclear Leucocytes

YUTAKA TAMURA and MAYUMI TANAKA

First Assay Division, National Veterinary Assay Laboratory, 1-15-1 Tokura, Kokubunji, Tokyo 185 (Japan)

(Accepted for publication 8 August 1986)

ABSTRACT

Tamura, Y. and Tanaka, M., 1987. Opsonic activiW of anti-flagellar serum against Clostridium chauvoei by mouse polymorphonuclear leucocytes. Vet. Microbiol., 14: 81-86.

The role of anti-flagellar serum against Clostridium chauvoei in phagocytosis by mouse poly- morphonuclear leucocytes was examined. Anti-flagellar serum markedly increased phagocytic rate against the flagellated strain Okinawa but not against a non-flagellated mutant (NFM) derived from the same strain, while anti-NFM serum increased the phagocytic rate against both strains. These results indicate that anti-flagellar serum exerts its protective effect by opsonic activity.

INTRODUCTION

The protective antigens of Clostridium chauvoei have been shown to exist on both cell wall (Chandler and Hamilton, 1975a,b) and flagella (Tamura et al., 1984). However, the mechanism by which immunization enhances the sur- vival of animals infected with C. chauvoei is still not fully understood.

Our previous report showed that specific anti-flagellar serum against C. chauvoei had a powerful protective effect, preventing bacterial growth in the liver of mice challenged i.m. (Tamura and Tanaka, 1984). However, no pro- tective effect was observed in mice with polymorphonuclear leucocytes (PMN) depleted by cyclophosphamide treatment.

The present study was undertaken to determine the opsonic activity of anti- flagellar serum against C. chauvoei using mouse PMN.

MATERIALS AND METHODS

Bacterial strains

The C. chauvoei used in this study was the Okinawa strain, which has been used for vaccine production and potency assays in Japan (Azechi et al., 1962 ).

0378-1135/87/$03.50 © 1987 Elsevier Science Publishers B.V.

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A non-flagellated mutant (NFM) FN-42 was isolated by treating strain Oki- nawa with N-methyl-N'-nitro-N-nitrosoguanidine and was characterized as described previously ( Tamura et al., 1983).

Animals

Forty female 6-week-old ddY mice were purchased from the Shizuoka Agri- culture Cooperative Association for Laboratory Animals, Hamamatsu, Japan. Two rabbits of both sexes, weighing about 3 kg, were obtained from the Sankyo Labo Service, Tokyo, Japan.

Antisera

Anti-flagellar serum was prepared in rabbits by immunization with partially purified flagella of strain Okinawa as described previously (Tamura and Ta- naka, 1984). To prepare anti-NFM serum, strain FN-42 was grown anaerobi- cally at 37 ° C for 18 h in a cysteine-liver infusion broth without glucose (Azechi et al., 1962). Formalin was then added at a final concentration of 0.5%. The bacterial cells were washed three times with Dulbecco's phosphate buffered saline (PBS), pH 7.4, adjusted to the optical density of McFarland No. 5, and injected i.v. into rabbits at 4-day intervals in volumes of 0.5, 1.0, 1.5 and 2.0 ml. The rabbits were bled 10 days after the final injection.

Tests for the serological properties of antisera

Tests for the serological properties of antisera were performed as described previously (Tamura and Tanaka, 1984). Formalinized whole cells or boiled cells prepared from the Okinawa strain were used as H antigen or 0 antigen, respectively. For the immobilization test, 2-fold serial dilutions of the serum were made in PBS. A volume of 0.3 ml of each serum dilution was gently mixed with 2.7 ml of molten cysteine-liver infusion agar medium containing 0.15% agar, and the Okinawa strain was stab-inoculated into each tube and incu- bated. The highest dilution of serum that inhibited the migration of the orga- nism was termed the immobilization titer of the serum. Values of the 50% effective dose (EDs0) of the serum were determined as follows. Groups of four mice received an i.p. injection of 0.5 ml of 2-fold serial dilutions of antiserum and after 6 h they were challenged i.m. with 2.3 × 102 spores of strain Okinawa suspended in 0.25 ml of 3% CaC12 solution (100 MLD). EDso values were cal- culated by the method of Spearman and K~irber (Finney, 1964 ).

Measurement of serum opsonic activity

Immune phagocytosis was performed in the peritoneal cavity of the mouse. Exudation of PMN was induced by i.p. injection with 2 ml of 0.2% sodium

TABLE I

Serological properties of antiserum to C. chauvoei

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Antiserum a Agglutination Immobilization titer b titer b

EDso( × 10 -3 ml) c

H 0

Flagella 163 840 < 20 1280 8.8 NFM 80 320 ,; 20 35.0

aPartially purified flagella of strain Okinawa or bacterial cells of non-flagellated mutant (NFM) FN-42 were used for preparation of anti-flagella or anti-NFM serum, respectively. bThe agglutination and immobilization tests were performed against strain Okinawa as described in the text. CEDso values were determined against strain Okinawa.

caseinate. Three hours after injection, mice were challenged i.p. with strains Okinawa or FN-42. At this time, approximately 80% of the peritoneal exudate cells were PMN. For opsonization, anti-flagellar serum or an t i -NFM serum were used. Antisera and normal rabbit serum were diluted 10-fold with Hank's balanced salt solution (HBSS) , and groups of five mice were injected i.p. with 0.5 ml of each sera or HBSS 1 h before challenge. Bacterial strains were grown anaerobically as described above. The bacterial cells were washed three times with HBSS, and 0.5 ml of bacterial suspension containing 9.0 × 101° bacteria was injected into the peritoneal cavity of the mouse. At various times after injection, peritoneal cells were collected by a microcapillary tube (Terumo Corp., Tokyo, Japan) and smeared on a glass slide. After drying, the cells were stained with Wright 's stain. The percentage of PMN ingesting bacteria was determined under a microscope for >/100 PMN per mouse.

RESULTS

Serological properties of the antisera tested

Table I shows the serological properties of the antisera tested. The H agglu- t inating titer of the anti-flagellar serum was 163 840, and tha t of the O agglu- t inat ing was < 20. On the other hand, the H agglutinating titer of the anti- NFM serum was 80, but the O agglutinating titer was 320. The immobilization titer in soft agar of these antisera was lower than the corresponding titers of H agglutination. However, it is clear tha t these titers reflect the presence of flagellar antibodies in the antisera. These results imply the possible roles of flagellar or somatic antibodies in phagocytosis by using such antisera.

The EDso value of anti-flagellar serum was significantly higher than tha t of

m

o o

~p

30

20

i0

0

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Strain Okinawa

a

3o 60 12o

Strain FN-42

0 30 60 120

Time after inoculation (min)

Fig. 1. Effect of anti-flagellar serum and anti-NFM serum to C. chauvoei in phagocytosis by mouse polymorphonuclear leucocytes. Vertical bars represent standard errors of means of five mice. C), anti-flagellar serum; Q, anti-NFM serum; A, normal rabbit serum; A, Hank's balanced salt solution.

ant i -NFM serum. Heating the anti-flagellar serum at 56 °C for 30 min did not significantly alter this EDso value.

Opsonic activity of anti-flageUar serum and anti-NFM serum

To examine the opsonic activity of the antisera, mice pretreated with anti- flagellar serum or ant i -NFM serum were challenged with either strains Oki- nawa or FN-42. The kinetics of opsonization are shown in Fig. 1. Effective opsonization by mouse P M N occurred after 30 min; the percentage of P M N ingesting bacteria at 30 min was therefore determined. The anti-flagellar serum and ant i -NFM serum supported phagocytosis of strain Okinawa by mouse PMN. A mean of 23% and 18% phagocytosis, respectively, was observed for strain Okinawa opsonized with anti-flagellar serum or ant i -NFM serum. Nor- mal rabbit serum and HBSS, however, were ineffective as an opsonin for strain Okinawa, resulting in approximately 10% phagocytosis, respectively. These results indicate that strain Okinawa was opsonized more effectively with anti- flagellar serum.

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On the other hand, mouse PMN phagocytized strain FN-42 opsonized with anti-NFM serum (26%) but not with anti-flagellar serum (10%). Strain FN- 42 opsonized with normal rabbit serum or HBSS was not phagocytized. Only 7% of normal rabbit serum-opsonized bacteria and 5% of HBSS-treated bac- teria were ingested. Thus, in contrast to mouse PMN phagocytosis of strain Okinawa, strain FN-42 phagocytized only in the presence of anti-NFM serum.

DISCUSSION

There is no definitive explanation of why bacterial flagella play an important role in the induction of immune resistance in animals. Previously, we suggested that the protective mechanism involves the prevention of the systemic spread of C. chauvoei by anti-flagellar serum, and the protective effects of this serum are exhibited only in the presence of PMN rather than macrophages (Tamura and Tanaka, 1984 ). The present study proved that the anti-C, chauvoei flagel- lar serum markedly increased the phagocytic rate against the flagellated Oki- nawa strain but not against strain FN-42, lacking flagella. These results, coupled with our previous observations, indicate that anti-flagellar serum exerts its protective effect by opsonic activity and that the opsonized C. chauvoei are eliminated by PMN.

It is known that opsonic adherence to phagocytes, which involves binding through IgG antibodies and complement, is of major importance in the host defence against systemic infection (Roitt, 1977). In a recent report, immuni- zation with the flagella of Vibrio cholerae decreased the number of bacteria attached to the mucosa of challenged animals (Guentzel et al., 1977; Yancey et al., 1979). Therefore, the protective mechanism by which bacterial flagella exert their effects in animals may consist of the prevention of either systemic or local infection by specific flagellar antibodies.

On the other hand, the anti-NFM serum was also found to increase phago- cytic rate against both strains Okinawa and FN-42. The result suggests that protective mechanism induced by cell wall of C. chauvoei ( Chandler and Ham- ilton, 1975a,b) is fundamentally the same as that by the flagella of the orga- nism. Further studies are necessary to elucidate where the major protective antigen is in the flagellum or the cell wall.

ACKNOWLEDGEMENT

We thank Dr. T. Sawada, National Veterinary Assay Laboratory, Tokyo, Japan, for review of the manuscript.

REFERENCES

Azechi, H., Seto, K., Mukawa, Y. and Suzuki, K., 1962. Studies on the potency test of blackleg vaccine. Jpn. J. Vet. Sci., 24: 267-275.

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Chandler, H.M. and Hamilton, R.C., 1975a. The preparation of immunogenic cell walls from a highly protective strain of Clostridium chauvoei. J. Gen. Microbiol., 88: 27-35.

Chandler, H.M. and Hamilton, R.C., 1975b. The protective antigenicity of protoplasts and sphaer- oplasts of a highly protective strain of Clostridium chauvoei. J. Gen. Microbiol., 88: 179-183.

Finney, D.J. (Editor), 1964. Statistical Methods in Biological Assay, 2nd edn., Charles Griffin, London, 524 pp.

Guentzel, M.N., Field, L.H., Eubanks, E.R. and Berry, L.J., 1977. Use of fluorescent antibody in studies of immunity to cholera in infant mice. Infect, Immun., 15: 539-548.

Roitt, I.M., 1977. Essential Immunology, 3rd edn., Blackwell Scientific Publications, Oxford- /London/Edinburgh/Melbourne, 136 pp.

Tamura, Y., Minamoto, N. and Tanaka, S., 1983. Isolation and characterization of non-flagellated and paralyzed flagellated mutants of Clostridium chauvoei. Jpn. J. Vet. Sci., 45: 417-420.

Tamura, Y. and Tanaka, S., 1984. Effect of antiflagellar serum in the protection of mice against Clostridium chauvoei. Infect. Immun., 43: 612-616.

Tamura, Y., Minamoto, N. and Tanaka, S., 1984. Demonstration of protective antigen carried by flagella of Clostridium chauvoei. Microbiol. Immunol., 28: 1325-1332.

Yancey, R.J., Willis, D.L. and Berry, L.J., 1979. Flagella-induced immunity against experimental cholera in adult rabbits. Infect. Immun., 25: 220-228.