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Mucosal immunisation with Clostridium botulinum type C 16 S toxoid and its non-toxic component

NAZIRA MAHMUT, KAORU INOUE, YUKAKO FUJINAGA, HIDEYUKI ARIMITSU, YOSHIHIKO SAKAGUCHI, LYNN HUGHES^*, ROBERT HIRST^*, TOM MURPHY, TAKAO TSUJI, TOSHIHIRO WATANABE, TOHRU OHYAMA, TADAHIRO KARASAWA, SHINICHIRO NAKAMURA, KENJI YOKOTA and KEIJI OGUMA

Department of Bacteriology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho Okayama 700-8558, Japan, *Department of Microbiology and Immunology, School of Biomedical Sciences, James Cook University, Townsville 4811, Australia, National Water Research Institute, 867 Lakeshore Road, Burlington, Ontario L7R 4A6, Canada, Department of Microbiology, Fujita Health University, School of Medicine, Toyoake, Aichi 470-11, Japan, Department of Food Science, Faculty of Bioindustry, Tokyo University of Agriculture, 196, Yasaka, Abashiri 099-2422, Japan and Department of Bacteriology, Graduate School of Medical Science, Kanazawa University, 13-1 Takara Machi, Kanazawa 920-8640, Japan

Corresponding author: Dr K. Oguma (e-mail: kuma{at}md.okayama-u.ac.jp).

Received 11 March 2002; revised version accepted 11 June 2002.

Clostridium botulinum types C and D produce a 16 S (500 kDa) toxin that is formed by conjugation of neurotoxin with a non-toxic component (nonTox). The amino acid sequences of type C and D nonTox components are almost identical. In a previous report it was proposed that nonTox is necessary for the effective absorption of the toxin from the small intestine. This suggested the hypothesis that mucosal immunity against nonTox in the small intestine might prevent the absorption of both C- and D-16 S toxins. The nonTox was purified from a mutant strain, (C)-N71, that does not produce neurotoxin. This nonTox or detoxified C-16 S toxin were mixed with adjuvant (a mutant form of heat-labile toxin of Escherichia coli), and inoculated into mice via the nasal or oral route, or both. The mice inoculated nasally four times with nonTox or toxoid produced high levels of antibodies (including IgA) against the immunogens, both in intestinal fluids and sera. When these nonTox-immunised mice were challenged orally with 2 and 20 oral minimum lethal doses (MLD) of C- or D-16 S toxins, the same results were obtained with both C and D; the mice survived after challenge with 2 MLD of either C or D but were killed by 20 MLD of either toxin although the time to death was significantly longer than in the control non-immunised mice. These results indicate that the local anti-nonTox antibodies reduce absorption of both C- and D-16 S toxins from the small intestine. The C-16 S toxoid-immunised mice showed similar behaviour with type D toxin challenge, probably due to the same mechanism, but were protected against 20 MLD of C-16 S toxin.

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