Serological method for identification of Vibrio parahaemolyticus from marine samples

Use of agglutination with antiserum against lateral flagella (H-agglutination) for the identification of Vibrio parahaemolyticus was studied. Sucrose-negative bacteria were isolated from seawater, and their characterization was carried out by traditional biological tests and slide agglutination with antiserum specific to lateral flagella of V. parahaemolyticus. Of 135 strains isolated, 78 were identified as V. parahaemolyticus by biological tests and were agglutinated with the above serum. Fifty-five strains did not agglutinate with the serum, and their biological characteristics were different from those of V. parahaemolyticus. Two strains also differed from V. parahaemolyticus in some biological characteristics but agglutinated with the antiserum. All clinically isolated V. parahaemolyticus strains also agglutinated with the above serum. These results suggest that our serological method is useful for the identification of V. parahaemolyticus, especially for samples in which there are many organisms related to V. parahaemolyticus, because many biological tests can be omitted.     

Differences between surface antigenic determinants of polar monotrichous flagella of Vibrio parahaemolyticus and of related species

Polar monotrichous flagella (M-flagella) of Vibrio parahaemolyticus have antigens in common with those of various species of Vibrio including V. cholerae and V. anguillarum, and of Beneckea, revealed by gel diffusion tests with flagelli monomers. However, antiserum against M-flagellin of V. parahaemolyticus did not agglutinate cells of V. cholerae and V. anguillarum, although it did agglutinate cells of V. parahaemolyticus. Agglutination tests after absorption of the antiserum with purified M-flagellar filaments or flagellin monomers and H-agglutination inhibition tests demonstrated that there are two different antigenic determinants in M-flagella as in lateral flagella. One is on the surface of the M-flagella (surface antigenic determinant, SA) and disappears or is buried in dissociated monomers. The other is inside the flagella (internal antigenic determinant, IA) and is exposed when the flagella are dissociated to flagellin monomers. SA of V. parahaemolyticus is different from those of V. cholerae and V. anguillarum, whereas the three species have a common IA.     

The lipopolysaccharide (R type) as a common antigen of Neisseria gonorrhoeae. II. Use of hen antiserum to gonococcal lipopolysaccharide in a rapid slide test for the identification of N. gonorrhoeae from primary isolates and secondary cultures.

An antiserum has been prepared in hens to R-type gonococcal lipopolysaccharide (LPS) and used in a simple slide-agglutination test for the identification of Neisseria gonorrhoeae. Anti-LPS serum agglutinated gonococcal cells representative of the four colony types of N. gonorrhoeae. Absorption of the antiserum with LPS removed the agglutinating activity. Secondary cultures (1120) were tested without observation of the colony type and all were agglutinated. No agglutination occurred with strains of Neisseria meningitidis, Neisseria lactamica, non-pathogenic Neisseria. Pseudomonas aeruginosa, Branhamella catarrhalis, or with species of lactobacilli and Acinetobacter. Cross-reactivity of the antiserum occurred with some streptococci. The anti-LPS serum was used to identify N. gonorrhoeae in primary isolates from the cervix, urethra, and pharynx. Of 251 gonococcal isolates tested, 249 were agglutinated by the antiserum, while all of the corresponding second cultures were agglutinated. The antiserum did not agglutinate N. meningitidis found in primary isolates from pharyngeal specimens. Anti-LPS hen serum should be useful for the rapid identification of N. gonorrhoeae in primary isolates or secondary cultures.     

Identification of Isolates of Clostridium perfringens Types C and D by Agglutination and Fluorescent-Antibody Methods

Agglutination and fluorescent-antibody methods were employed for screening Clostridium perfringens types C and D from 393 isolates of this organism. All of 50 strains which were isolated in Japan and were agglutinable with an antiserum prepared against a stock strain of type C no. 3182 toxigenically belonged to type C, but the antiserum showed no cross-agglutination with any of type C strains isolated in Denmark. All of the latter strains, however, were agglutinated by an antiserum prepared against a Danish strain, CWC11. Of 64 strains, showing heat-labile agglutinability by type D antiserum L9, 22 strains were toxigenically identified as type D strains which can be divided into three groups by the heat-stable antigens; no strains which were L-agglutination-positive but O-agglutination-negative were epislon-toxigenic. All of 13 strains, the heat-stable antigen of which was agglutinable by a type D antiserum VX81, were toxigenically type D strains. The results of fluorescent-antibody tests were almost in agreement with those of agglutination test with type C strains and completely with those of the O-agglutination test with type D strains. No beta-, epsilon- or delta-toxigenicity could be demonstrated in strains which were not agglutinated by our test sera for types C and D strains. Further examination of cultural properties of Japanese and Danish type C strains revealed that the two groups were considerably different in urease production, capsule formation, and delta- and alpha-toxigenicities.     

Lateral flagellar antigen of Vibrio alginolyticus and Vibrio harveyi: existence of serovars common to the two species

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 between 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.     

Production of pili on Vibrio parahaemolyticus

Electron microscopic examination showed that all strains of Vibrio parahaemolyticus examined had pili on their surface when the organism was grown on marine agar at 28 degrees C for 6-12 h. The pili were morphologically stable on heat treatment at 60 degrees C for 10 min, but both the lateral and polar flagella possessed by this organism were labile. No immunological cross-reactivity between pili of enterotoxigenic Escherichia coli and Vibrio cholerae non-01 and those of V. parahaemolyticus was observed.     

Isolation of the polar and lateral flagellum-defective mutants in Vibrio alginolyticus and identification of their flagellar driving energy sources.

Vibrio alginolyticus has two types of flagella (polar and lateral) in one cell. We isolated mutants with only a polar flagellum (Pof+ Laf-) or only lateral flagella (Pof- Laf+). Using these mutants, we demonstrated that the energy sources of the lateral and polar flagellar motors in V. alginolyticus are H+ and Na+ motive forces, respectively, as in the related species V. parahaemolyticus.     

An antiserum against a glycoprotein functional in flagellar adhesion between Chlamydomonas eugametos gametes

Chlamydomonas eugametos gametes agglutinate sexually by their flagellar surfaces. The agglutination factor on mating type minus (mt^-) gametes is thought to be a glycoprotein named PAS-1.2. To test this idea, an antiserum was raised against purified PAS-1.2., which reacted with isolated PAS-1.2 (immunoprecipitation tests) and blocked the ability of isolated PAS-1.2 to induce sexual twitching in mt ⁺ gametes. When tested with living cells, the antiserum specifically agglutinated mt ^- gametes and induced a reaction resembling twitching. Mt ⁺ flagella were shown to bind the antiserum (indirect immunofluorescence) but much less than mt ^- gametes. Mt ^- gametes pretreated with Fab fragments of the antiserum were unable to reproduce sexually, while treated mt ⁺ gametes were unaffected. This effect presumably results from the ability of the serum to block mt ^- sexual agglutination, for mt ^- isoagglutinin was completely inactivated by the serum, while mt ⁺ isoagglutinin was unaffected. It is therefore argued that PAS-1.2 is the in vivo mt ^- agglutination factor. However it is shown that the antiserum was able to react in vitro not only with PAS-1.2 but with several other proteins in both mt ^- and mt ⁺ flagella.Abbreviations SDS sodium dodecyl sulphate - PAS periodic acid-Schiff - GTC guanidine thiocyanate - mt ^+/- mating type plus or minus - PBS phosphate buffer-saline - Fab univalent antibody fragment The investigations were supported by the Foundation for Fundamental Biological Research (BION), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (Z.W.O.)     

Serological Studies of Clostridium botulinum Type E and Related Organisms II. Serology of Spores

Pure spore antigens for the immunization of rabbits were prepared by enzymic digestion of vegetative components and separation of the cleaned spores in polyethylene glycol. Spore antisera were prepared to strains representative of toxigenic Clostridium botulinum type E; nontoxigenic boticin E-producing variants; nontoxigenic nonproducers of boticin E; nontoxigenic "atypical" strains, which differ somewhat from C. botulinum type E in their physiology; C. botulinum types A and B; and C. bifermentans. They were tested against these and additional strains representative of the above groups, other types of C. botulinum, and other Clostridium species. There was no evidence of agglutination of flagellar or somatic antigens of vegetative cells by these antisera. Agglutination and agglutinin absorption tests showed common antigens among toxigenic type E strains and nontoxigenic variants, both producers and nonproducers of boticin E. Some nontoxigenic "atypical" strains varied in their ability to be agglutinated by type E antisera, and others did not agglutinate at all. Of those atypical strains that were not agglutinated, one was agglutinated by C. bifermentans antiserum. Antisera prepared against C. botulinum types A and B and C. bifermentans did not agglutinate the spores of type E or its variants nor share antigens common to each other. Similarly, antisera to type E, its nontoxigenic variants, and nontoxigenic atypical strains did not agglutinate other C. botulinum types or any other Clostridium species investigated.     

Antigen sharing of Salmonella typhi non-flagellar proteins with other salmonellae and some shigellae and Escherichia coli

Cathodal moving protein components were identified in agarose gel electrophoresis of the Veronal buffer extract of a non-motile strain of S. typhi (8393, Colindale). Rabbit antiserum was raised against these cationic proteins; it had both agglutinating and precipitating activity. A total of 80 salmonella strains belonging to serogroups A, B, C1, C2, D, E1 and E2 including 26 S. typhi and 10 S. paratyphi A were tested against this antiserum in a slide agglutination test; all strains were agglutinated. Among 94 other bacterial strains tested, the antiserum agglutinated all 16 strains of Shigella flexneri, 2 of 5 Shigella sonnei, 5 of 34 E. coli and 1 of 8 Citrobacter species. These results show that there is antigenic sharing of the non-flagellar proteins of S. typhi with many other salmonellae as well as with some shigellae and E. coli.     

Detection and identification of Vibrio species using whole-cell protein pattern analysis

Outbreaks of foodborne diseases associated with Vibrio species such as V. parahaemolyticus, V. vulnificus, and V. cholerae frequently occur in countries having a dietary habit of raw seafood consumption. For rapid identification of different Vibrio species involved in foodborne diseases, whole-cell protein pattern analysis for 13 type strains of 12 Vibrio species was performed using SDS-PAGE analysis. Pathogenic Vibrio species such as V. parahaemolyticus, V. vulnificus, V. cholerae, V. alginolyticus, V. fluvialis, and V. mimicus were included in the 12 Vibrio species used in this study. Each of the 12 Vibrio species showed clearly specific band patterns of its own. Two different strains of V. parahaemolyticus showed two different SDS-PAGE wholecell protein patterns, giving the possibility of categorizing isolated strains in the same V. parahaemolyticus species into two subgroups. The 36 Vibrio isolates collected from sushi restaurants in Busan were all identified as V. parahaemolyticus by comparing their protein patterns with those of Vibrio type strains. The identified isolates were categorized into two different subgroups of V. parahaemolyticus. The whole-cell protein pattern analysis by SDS-PAGE can be used as a specific, rapid, and simple identification method for Vibrio spp. involved in foodborne diseases at the subspecies level.     

Identification with the aid of various serological reactions of L forms of streptococci isolated from the live organism]

Growth inhibition, agglutination, precipitation, and passive hemagglutination tests were used for the identification of the L-forms of streptococci isolated from the organism of experimental rabbits both after the infection with the L-forms of streptococci and with the streptococci of group A. The tests were positive not only with the antiserum of homologous, but also of heterologous strains of the L-form of streptococcus, group A. The L-form cultures isolated from the experimental animals failed to differ from the laboratory strain of the L-forms of streptococcus, group A, by serological properties.     

Differences in the API 20E biochemical patterns of clinical and environmental Vibrio parahaemolyticus isolates

Genetic differences in clinical and environmental strains of Vibrio parahaemolyticus have been widely used as criteria in identifying pathogenic isolates. However, few studies have been carried out to assess the differences in biochemical characteristics of V. parahaemolyticus isolates from human and environmental sources. We compared the biochemical profiles obtained by the characterization of V. parahaemolyticus isolates from human infections and the marine environment using the API 20E system. Environmental and clinical isolates showed significant differences in the gelatin and arabinose tests. Additionally, clinical isolates were correctly identified according to the API 20E profile using 0.85% NaCl diluent, but they presented nonspecific profiles with 2% NaCl diluent. In contrast, use of 2% NaCl diluent facilitated correct identification of the environmental isolates. Clinical isolates showed significant differences in up to five biochemical tests with respect to the API 20E database. The API 20E system is widely used in routine identification of bacteria in clinical laboratories, and this discrepancy in an important number of biochemical tests may lead to misidentification of V. parahaemolyticus infection.     

Scanning electron microscope observation of the swarming phenomenon of Vibrio parahaemolyticus.

Scanning electron microscopy was used to study the production of lateral flagella and the swarming phenomenon in Vibrio parahaemolyticus. Differences in the size and diameter of the sheathed, polar flagellum and lateral flagella were apparent in these preparations. Swarming of V. parahaemolyticus was found to be similar to the swarming of Proteus spp. in that swarm cells which are heavily flagellated and elongated are formed.     

霍乱弧菌和副溶血弧菌分离株的gyrB基因系统发育分析

依据gyrB基因部分编码序列构建系统发育树以分类和鉴别霍乱弧菌和副溶血弧菌,并探讨其种系发生关系。扩增并测序13株霍乱弧菌、8株副溶血弧菌、2株嗜水气单胞菌及1株类志贺邻单胞菌的gyrB基因(编码DNA促旋酶B亚单位)序列,并采用距离法与最大似然法构建系统发育树。两种方法所构建的树结构完全一致,霍乱弧菌、副溶血弧菌、嗜水气单胞菌及类志贺邻单胞菌各自形成一个独立的簇。其中,霍乱肠毒素基因(ctxA)阳性的霍乱弧菌(8株O139群与2株O1群ElTor型)聚类成一分枝;3株副溶血弧菌临床株(1株2002年流行株,2株2004年分离株)与1日本菌株及2001年1株自环境分离的毒力株聚类。系统发育分析靶分子gyrB基因可以良好区分上述4种常见病原菌。产毒O139群霍乱弧菌与产毒O1群ElTor型霍乱弧菌关系密切。副溶血弧菌环境毒力株与本地区临床主要流行株在系统发育关系上较为接近,可能是潜在的致病菌。     

Adsorption kinetics of laterally and polarly flagellated Vibrio.

The adsorption of laterally and polarly flagellated bacteria to chitin was measured, and from the data obtained, a modified Langmuir adsorption isotherm was derived. Results indicated that the adsorption of laterally flagellated Vibrio parahaemolyticus follows the Langmuir adsorption isotherm, a type of adsorption referred to as surface saturation kinetics, when conditions are favorable for the production of lateral flagella. When conditions were not favorable for the production of lateral flagella, bacterial adsorption did not follow the Langmuir adsorption isotherm; instead, proportional adsorption kinetics were observed. The adsorption of some polarly flagellated bacteria exhibited surface saturation kinetics. However, the binding index (the product of the number of binding sites and bacterial affinity to the surface) of polarly flagellated bacteria differed significantly from that of laterally flagellated bacteria, suggesting that polarly flagellated bacteria adsorb to chitin by a different mechanism from that used by the laterally flagellated bacteria. From the results of dual-label adsorption competition experiments, in which polarly flagellated V. cholerae competed with increasing concentrations of laterally flagellated V. parahaemolyticus, it was observed that laterally flagellated bacteria inhibited the adsorption of polarly flagellated bacteria. In contrast, polarly flagellated bacteria enhanced the adsorption of V. cholerae. In competition experiments, where V. parahaemolyticus competed against increasing concentrations of other bacteria, polarly flagellated bacteria enhanced V. parahaemolyticus adsorption significantly, whereas laterally flagellated bacteria only slightly enhanced the process. The direct correlation observed between surface saturation kinetics, the production of lateral flagella, and the ability of laterally flagellated bacteria to inhibit the adsorption of polarly flagellated bacteria suggests that lateral flagella represent a component of bacterial structure that is important in the adsorption of laterally flagellated bacteria to surfaces. A model for adsorption events of laterally flagellated bacteria is proposed, based on the evidence presented.     

Occurrence and characteristics of agglutination of Vibrio cholerae by serum from the eastern oyster, Crassostrea virginica.

Cell-free hemolymph (serum) of the eastern oyster, Crassostrea virginica, agglutinated Vibrio cholerae, including all O1 serovars and biovars. Seventy-nine other strains of bacteria, including 14 genera and 26 species, were not agglutinated. The A, B, and C factors of O1 antigen were not involved in agglutination. Bacterial agglutinating (BA) activity was demonstrated for oysters inhabiting different environments of the U.S. Atlantic and Gulf coasts. Oyster serum BA titers showed high individual variation. The serum component(s) involved in BA was inhibited by 80 degrees C heat, pronase, EDTA, mucin, and fetuin treatments. N-Acetylneuraminic acid (10 mg/ml) weakly inhibited BA activity. Ligands of V. cholerae were sensitive to neuraminidase and resistant to 80 degrees C and pronase. High salinities (24 and 30%) enhanced BA. Cross-adsorption tests with V. cholerae and human O+ erythrocytes indicated that BA and hemagglutinating activities may involve different serum components. These results imply that the ecology of V. cholerae in C. virginica is influenced by agglutinating activity of oyster serum.     

Occurrence and characteristics of agglutination of Vibrio cholerae by serum from the eastern oyster, Crassostrea virginica

Cell-free hemolymph (serum) of the eastern oyster, Crassostrea virginica, agglutinated Vibrio cholerae, including all O1 serovars and biovars. Seventy-nine other strains of bacteria, including 14 genera and 26 species, were not agglutinated. The A, B, and C factors of O1 antigen were not involved in agglutination. Bacterial agglutinating (BA) activity was demonstrated for oysters inhabiting different environments of the U.S. Atlantic and Gulf coasts. Oyster serum BA titers showed high individual variation. The serum component(s) involved in BA was inhibited by 80 degrees C heat, pronase, EDTA, mucin, and fetuin treatments. N-Acetylneuraminic acid (10 mg/ml) weakly inhibited BA activity. Ligands of V. cholerae were sensitive to neuraminidase and resistant to 80 degrees C and pronase. High salinities (24 and 30%) enhanced BA. Cross-adsorption tests with V. cholerae and human O+ erythrocytes indicated that BA and hemagglutinating activities may involve different serum components. These results imply that the ecology of V. cholerae in C. virginica is influenced by agglutinating activity of oyster serum.     

Taxonomy and ecology of brucellosis pathogens isolated from Muridae in the northern foothills of the Caucasus mountains. I. Cultural and biochemical properties of Brucella isolated from Muridae

The results of the study of 65 Brucella strains isolated from myomorphous rodents in the Northern Caucasus are presented. The study was made with the aim of finding out additional characteristics for the identification of these strains. Using the main tests recommended by the FAO/WHO Subcommittee on the Taxonomy of Brucella, as well as some additional tests, we have revealed that the strains under study are very similar to B. suis. At the same time their capacity for agglutination with anti-melitensis monospecific serum, their high sensitivity to pyronin B, safranine T and gentian violet, their low urease activity and their oxidizing activity in respect to L-alanine, L-asparagine, L-glutamic acid, L-arginine, DL-ornithine, DL-citrullin and L-livin make it possible to consider them the fifth independent biotype of B. suis.