Cells of Vibrio parahaemolyticus treated with Formalin agglutinated with anti-lateral flagella antiserum. On the basis of agglutination tests, antigens of lateral flagella were divided into three groups, HL1, HL2, and HL3. However, in gel diffusion tests, flagellins prepared from strains belonging to different groups showed a common antigenicity. It is assumed that these results are due to the existence of two distinct antigenic determinants in the lateral flagella. One of them exists on the surfaces of the flagella and is responsible for H-agglutination, and the other exists inside the flagella and is exposed when the flagella are solubilized to flagellin monomers.Cells of Vibrio parahaemolyticus grown in liquid media are monotrichous, having only a polar flagellum (M-flagellum), whereas those grown on solid media possess lateral flagella (b-flagella) in addition to an M-flagellum (1-3, 16, 17). Previously, we demonstrated the antigenic difference between these two kinds of flagella (9-11). Although b-flagella were easily removed from the cells by gentle shaking, they could be fixed on the cells by Formalin treatment. Therefore, H-agglutination could be observed when the Formalin-treated cell suspension was mixed with anti-L-flagella antiserum.During the course of our study, in agglutination tests it was observed that V. parahaemolyticus could be divided into three groups by Hagglutination, although the purified flagellins from cells belonging to these three different groups formed a common precipitin line in gel diffusion tests. In this paper we analyze the antigenicity of the b-flagella of V. parahaemolyticus and report the existence of two distinct antigenic determinants in flagella.Cells of V. parahaemolyticus grown on MMOF agar medim (10) at 25°C for 16 to 20 h were treated with a 3% Formalin solution containing 3% sodium chloride to fix the b-flagella on the cells. Absorbance at 600 nm of the fixed cell suspension was adjusted to 0.7 by a Shimadzu Spectronic 20 photometer. The suspension was mixed with the same volume ofdiluted anti-b-flagella antiserum and was incubated at 50°C. After 2 to 4 h of incubation, typical Hagglutination was observed. Antisera were prepared by immunizing rabbits with purified fla-gella (10). These anti-b-flagella antisera were specific to b-flagella and did not contain antibody against M-flagellum or other cell components. Table 1 shows that cells of V. parahaemolyticus WP-1 (RIMD 2210086), OV-19 (RIMD 2210033), and OV-24 (RIMD 2210036) agglutinated with anti-L-flagella antiserum prepared from homologous strains but not with that prepared from heterologous strains. A total of 105 out of 110 strains tested agglutinated with one ofthese three antisera. Microscopic observation of cells stained by the Leifson method (7) showed that cells that did not agglutinate with any antisera did not possess b-flagella.It was found that there was a certain relationship between H-agglutination and 0-antigenicity of V. parahaemolyticus. Strains of V. parahaemolyticus belonging to the 02 group ag...

The antigenicity of lateral (L-) flagella of two marine vibrios, Vibrio alginolyticus and V. harveyi, was studied, and the two species were found to have common antigenicity of their flagella. Antisera against L-flagella were prepared by immunizing rabbits with highly purified L-flagellar filaments. H-Agglutination tests with the anti-L-flagella antisera showed that four H-serovars existed in these species and that two of them were shared by the two species. Cross reactivity be tween H-serovars of these two species and other vibrios having lateral flagella, such as V. parahaemolyticus, V. campbellii, V. proteus, or V. fluvialis, was not observed in the H-agglutination test, although partial common antigenicity was observed in the gel diffusion test with flagellin monomers. These observations suggest that surface antigenic determinants of the lateral flagella of V. alginolyticus and V. harveyi are specific to these two species but internal antigenic determinants buried in the flagellar filaments are partially shared with other vibrio species.Some species of genus Vibrio have two kinds of flagella, lateral flagella (Lflagella) and polar monotrichous flagella (M-flagella) (1,4,20). Cells grown in liquid media possess only one M-flagellum on one end of each rod cell, whereas cells grown on agar media have both kinds of flagella on one cell. M-Flagella and L-flagella serve a locomotive function in a liquid environment and on a solid surface, respectively (19). We demonstrated that the antigenicity of these two flagella of V. parahaemolyticus are different from each other, and that two different antigenic determinant groups, namely a surface antigenic determinant (SA) and an internal antigenic determinant (IA), exist on both flagella (13,16,18). SA is located on the surface of flagellar filaments, and is responsible for H-agglutination.IA exists inside the flagella and is exposed only when the flagellar filaments are dissociated into flagellin monomers. IA is responsible for the gel diffusion reaction with flagellin.L-Flagellar antigens of V. parahaemolyticus are divided into three serovars, HL-1, HL-2, and HL-3, by means of SA. There is no cross reactivity between the SAs of L-flagella of the three serovars, and cross reactivity with other vibrios was not observed (16). Therefore, the SAs of L-flagella of V. parahaemolyticus are species 173

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The behavior of a surface antigenic determinant (SA) of lateral flagella of Vibrio parahaemolyticus was studied. SA, located on the surface of flagellar filaments, is responsible for H-agglutination and specific for serotype. Results of H-agglutination inhibition tests demonstrated that SA could not be detected on the flagellin molecule when the flagellar filaments were dissociated to flagellin monomers by heating or treatment with urea, sodium dodecyl sulfate, HCl, or acetone, although SA could be detected on short flagellar fragments obtained by milder heat treatment. When flagellar filaments were reconstituted from flagellin monomers, SA was detected on the surface of the filaments. These results suggest that SA is buried in the flagellin molecule with dissociation of flagellar filaments to flagellin monomers or steric configuration of SA itself is altered to a different form which cannot react with the responsible antibody.