Agglutination Typing of Vibrio anguillarum Isolates from Diseased

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1984, p /84/ $02.00/0 Copyright ) 1984, American Society for Microbiology Vol. 47, No. 6 Agglutination Typing of Vibrio anguillarum Isolates from Diseased Fish and from the Environment JENS L. LARSEN'* AND STIG MELLERGAARD2 Institute of Hygiene and Microbiology,1 and Fish Disease Laboratory,2 Danish Institute for Fishery and Marine Research, The Royal Veterinary and Agricultural University, DK-1870 Copenhagen V, Denmark Received 14 November 1983/Accepted 21 February 1984 Agglutinating activity was widely distributed among 101 Vibrio anguillarum strains of different origin and three Vibrio ordalii strains from salmonids. The spectrum of cells which were agglutinated comprised yeast cells and human (type 0), poultry, guinea pig, and trout erythrocytes, whereas ovine, bovine, and tanned bovine erythrocytes were not affected. Mannose-sensitive hemagglutination, mannose-resistant hemagglutination, and non-agglutinating strains were recognized. The three V. ordalii strains showed mannoseresistant hemagglutination, whereas V. anguillarum exhibited either mannose-sensitive hemagglutination or was non-agglutinating. Among V. anguillarum, sensitivity to D-galactose and L-fucose occurred sporadically. An agglutination typing scheme was developed for strains of V. anguillarum based on the agglutination pattern of human, poultry, guinea pig, and trout erythrocytes and yeast cells. Eight different agglutination types (A through H) were defined. The distribution of these types among fish pathogenic and environmental V. anguillarum strains were studied. The application of the typing scheme in ecological and epidemiological studies and for preventive medical purposes is discussed. Vibrio anguillarum is the most important fish pathogenic bacterium in the marine environment (3), being responsible for the vibriosis syndrome. In acute cases, a hemorrhagic septicemia is observed, and ulcers are predominant in more protracted cases (14). Until recently, very little information existed on the pathogenesis of vibriosis (17). The discovery of a specific plasmid class in virulent V. anguillarum strains (8, 9) and a hemagglutinating ability of fish pathogenic Vibrio strains (25) has increased the knowledge of vibriosis, although the biological expression of the plasmids is unknown. V. anguillarum is a member of the indigenous flora in coastal waters (22, 23; D. A. West, Ph.D. thesis, University of Kent, Kent, England, 1980), for which adhesion to solid surfaces is a necessity for survival and multiplication (27). Attachment (adhesion) is an important virulence factor for many pathogenic bacteria (16) and is known to be the initial step in many infections. Adherence is mediated by surface structures called adhesins or colonization factors (4, 16, 18, 20). Receptors corresponding to these adhesins have been identified on host epithelial cells. Such receptors frequently contain oligosaccharides (4, 16, 21). A simple method, the hemagglutination test, for studying the adhesive properties of bacterial cells was introduced by Constable (6) and Duguid and Gillies (11) and has been further developed and intensively used during the last few years (13, 15). A method for typing V. anguillarum by the agglutination pattern was adapted and used in the study of V. anguillarum strains originating from diseased fish and the environment in addition to certain reference strains. Three V. anguillarum biotype 2 strains named Vibrio ordalii (24) were used for comparison. MATERIALS AND METHODS Organisms. A total of 101 V. anguillarum and 3 V. ordalii strains were investigated. All organisms except 16 reference strains were isolated in our laboratory. A total of 42 strains were isolated in pure culture from pronephros of diseased * Corresponding author fish as follows: cod (Gadus morhua), 10; eel (Anguilla anguilla), 11; rainbow trout (Salmo gairdneri), 18; and turbot (Scophtalmus maximus), 3. The 46 environmental strains originated from mucus (6 strains) and feces (7 strains) of cod, invertebrates (11 strains), plankton (3 strains), seawater (10 strains), and sediment (9 strains). The mucus and feces strains were isolated from pools of mucus and feces of healthy cod captured in various Danish coastal areas. The invertebrate isolates originated from crab (Carcinus maenas), snails (Littorina littorica), and mussels (Mytilus edulis). Strains from plankton, seawater, and sediment along with those from the invertebrate and cod were isolated at the same sites. A register of reference strains was requested for this study (Table 1). The isolated V. anguillarum strains were maintained on stock culture agar (Difco Laboratories) slants at 4 C and had only been submitted to one passage before use. Preparation of organisms, erythrocytes, and yeast cells. The strains investigated were cultivated on calf blood agar (blood agar base; Difco) with citrate added to stabilize calf blood. The inoculated blood agar plates were incubated aerobically for 24 h at 25 C. Surface growth was harvested with phosphate-buffered saline (PBS), ph 7.4, after 24 h of incubation. The density of the bacterial suspension was subsequently adjusted to ca. 2.5 x 109 cells per ml by spectrophotometrical calibration (optical density at 590 nm, 0.290). Human type 0, bovine, ovine, poultry, guinea pig, and rainbow trout blood were used in the hemagglutination assays. The blood was collected by venipuncture and stored for not more than 1 week in Alsever solution (2) at 4 C. Before use, the blood was washed three times in PBS, and a 3% (vol/vol) suspension in PBS was prepared. For the test with tanned bovine erythrocytes, the washed erythrocytes were treated with 0.003% (vol/vol) tannic acid for 10 min at 37 C (5) and washed twice in PBS before a 3% (vol/vol) suspension in PBS was prepared. Baker's yeast (Saccharomyces cerevisiae) was suspended and washed three times in PBS, and finally a 3% (vol/vol) suspension in PBS was prepared.

2 1262 LARSEN AND MELLERGAARD APPL. ENVIRON. MICROBIOL. TABLE 1. Reference strains of V. anguillarum and V. ordalii Strain Origin and location Source V. anguillarum (biotype 1) NCMB 6 Gadus callaris, Denmark National Collection of Marine Bacteria NCMB 407 Pleuronectes platessa, Scotland National Collection of Marine Bacteria ATCC Salmo trutta, Scotland American Type Culture Collection ATCC Gadus callaris, Denmark American Type Culture Collection L.S Oncorhynchus tschawytscha, U.S.A. J. L. Fryer Oncorhynchus kisutsch, Canada K. A. Johnson Oncorhynchus kisutsch, Canada K. A. Johnson PT 24 Anguilla japonicus, Japan T. Aoki and K. Muroga PT 493 Plecoglossus altivelis, Japan T. Aoki and K. Muroga PT 213 Plecoglossus altivelis, Japan T. Aoki and K. Muroga PB 15 Plecoglossus altivelis, Japan T. Aoki PB 28 Plecoglossus altivelis, Japan T. Aoki ET 1 Plecoglossus altivelis, Japan T. Aoki V. ordalii (V. anguillarum biotype 2) 241-S Oncorhynchus nerka, U.S.A. M. H. Schiewe DF3K Oncorhynchus kisutsch, U.S.A. M. H. Schiewe M.S.C.2-75 Oncorhynchus kisutsch, U.S.A. J. L. Fryer Agglutination test. The agglutination assays were performed by mixing 20,ul of a bacterial suspension and 20 p.l of an erythrocyte or yeast cell suspension. As a control a 20-,u erythrocyte suspension or a yeast cell suspension was mixed with 20 pul of PBS. The agglutination test was performed on a squared transparent glass plate on which the agglutination of different strains with different cells could be cross-examined. The glass plate was continuously rocked during the procedure, and reading was carried out after 10 min and evaluated as positive or negative. Agglutination inhibition. Solutions (1%) of D-mannose, D- galactose, and L-fucose were used in the inhibition test. A 20-pl amount of a bacterial suspension was mixed with a 20- pul carbohydrate solution, and after 30 s of reaction time, a 20-plI erythrocyte or yeast cell suspension was added. For control the carbohydrate solution was replaced by 20 pi of PBS. The control and the three different carbohydrate solutions were tested simultaneously to assess inhibition. Sensitivity to sugar inhibition was recorded when agglutination was markedly reduced or absent. To confirm mannose-resistant hemagglutination (MRHA), the method of Duguid and coworkers (12) was adapted. Influence of temperature, ph, and NaCl concentration on agglutinating capacity. Blood agar base (Difco) without addition of blood was used in these experiments to avoid hemolytic phenomena due to unphysiological ph and NaCl concentrations. Two broad-spectrum agglutinating strains, NCMB 6 and 1347/1 (a cod isolate), were selected for these studies, and only yeast cell agglutination was tested. Bacteria grown on blood agar base under the following conditions were tested: 5, 10, 15, 20, 25, and 30 C growth temperature with blood agar base prepared in 0.1 M phosphate buffer at ph 6, 7, and 8 and incubated at 25 C and BAB with added NaCl to a final concentration of 1, 2, 3, and 4%. Twofold dilutions of the standard bacterial density (2.5 x 109) were tested, and the reciprocal value of the greatest dilution giving a positive reaction was recorded as the agglutination titer (AT). MAD and AP. Strains representing each of the different agglutination types were tested to estimate the minimum agglutinating dose (MAD) and agglutinating power (AP). Preparation of the bacteria was performed as described above, and the bacterial density was spectrophotometrically adjusted to ca. 8.0 x 1010 cells per ml. Serial twofold dilutions of this suspension were tested for agglutinating activity. MAD was defined as the smallest bacterial concentration giving visible agglutination within 10 min. AP was calculated as 101 l/mad according to Duguid and Gillies (11). Stability of agglutination capacity. Strains representing the most broad-spectrum agglutinating strains (A through C) were passed 60 times in tryptic soy broth (Difco) with 1% NaCl. After 20, 30, 40, 50, and 60 passages the bacteria were plated on blood agar, and AT was estimated as described above. RESULTS Influence of temperature, ph, and salinity. The optimal conditions for agglutinin production appear to be between 20 and 25 C, whereas the selected ph values and NaCl concentrations were of minor importance. In the following experiments, 25 C was chosen for bacterial growth to facilitate comparisons to previous reports (26). Agglutination typing of V. anguillarum (biotype 1 and 2) with erythrocytes and yeast cells tested in the presence and absence of carbohydrates with special emphasis on D-mannose. The V. anguillarum strains were found to exert mannosesensitive hemagglutination (MSHA) or to be non-agglutinable, whereas the three V. ordalii strains showed MRHA. As there was a pronounced difference in the hemagglutination patterns of V. anguillarum, it was decided to differentiate the strains into agglutination types with respect to the spectrum of cells agglutinated. With the four species of erythrocytes and the yeast cells, 32 possible types exist. However, the present strains could be grouped in six main types (A through F), a non-agglutinating type (G), and a group consisting of sporadically occurring types (H) (Table 2). Naturally, another collection of strains might necessitate an expansion of the system. Distribution of agglutination types among V. anguillarum strains of different origin. It is evident that there is a certain diversity in the agglutination pattern among the different species of fish and the different environmental strains (Table 3). All agglutinating strains agglutinated yeast cells. The erythrocytes from guinea pigs and poultry were the most common agglutinated blood cells (70 and 59%, respectively),

3 VOL. 47, 1984 TABLE 2. Agglutination of V. anguillarum and V. ordalii strains Erythrocytes Agglutination type of - Yeast strain Human Poultry Guinea Rainbow cells pig trout V. anguillaruma A B C D E F _ H V. ordaliib MSC S and DF3K C a MSHA agglutination types. b MRHA agglutination. C MSHA. whereas agglutination of human and trout erythrocytes was noticed among 29 and 24%, respectively. D-Mannose-sensitive agglutination was predominant, whereas D-galactose (15 strains) and especially L-fucose (2 strains) sensitivity occurred infrequently. Single outbreaks of vibriosis among rainbow trout and eels were found to be caused by specific agglutination types, but generally, the distribution was rather accidental. AT, MAD, and AP. Table 4 presents the AT, MAD, and AP of representative strains from each agglutination type. A tendency to decrease in agglutinating capacity from type A to G was noted. The agglutinating activity to the single erythrocyte type, however, shows a high degree of specificity. A titer of 32 is positive under standard conditions and corresponds to a bacterial cell concentration of 2.5 x 109. Since the D-mannose-sensitive agglutination was predominant, the inhibitive effect of this carbohydrate was examined. The minimum D-mannose-inhibiting concentration was found to be in the range of 0.9 to 1.7 mm. Stability of agglutination capacity. After 20 passages, a spontaneous loss of agglutination of specific species of erythrocytes was observed in some strains. But the main feature was that strains which showed no loss of agglutina- TABLE 3. Distribution of the agglutination types among fish pathogenic, environment, and reference strains of V. anguillarum Strain Distribution of agglutination types: A B C D E F G H Fish pathogenic Cod Eel Rainbow trout Turbot Environmental Feces, cod Mucus, cod Invertebrates Plankton Seawater Sediment Reference AGGLUTINATION TYPING OF V. ANGUILLARUM 1263 TABLE 4. AT, MAD, and AP of V. anguillarum strains representing the different agglutination types Aggluti- Cell nation Strain type' AT MAD AP type A 6198 E H x x 102 P x x 102 G x x 102 T x x 102 Y x x 102 B 13295/2 H x x 101 P x x 102 G x x 10' T x Y x x 102 C 1347/1 H x 10'0 5.0 P x x 102 G x x 102 T x x 102 Y x x 102 D 1310F/2 H x 10'0 2.5 P x x101 G x x 10' T x x 10l Y x x 102 E 1702/1 H x P x G x x 10 T x 10'0 5.0 Y x x 102 F H x P x x 10' G x x lo T x 10'0 1.3 Y x x 10' G a H, Human type 0; P, poultry; G, guinea pig; T, trout; and Y, yeast. tion after 20 passages maintained their stability throughout all 60 passages. DISCUSSION The ability to agglutinate yeast cells and erythrocytes from different animals is widely distributed among V. anguillarum strains. Both fish pathogenic and environmental strains possessed agglutinating properties. Typing of bacterial species with respect to their erythrocyte- and yeast cellagglutinating pattern has been performed. Thus, Evans and co-workers (13) defined seven hemagglutinating types in Escherichia coli based on both hemagglutination spectrum and MRHA and MSHA. Since the surface structures (fimbriae) of E. coli are very specific and of pathogenic importance, these have been incorporated in serotyping, which includes 0:, K:, H:, and F: antigens (10). The three V. ordalii strains exerted MRHA, whereas V. anguillarum showed MSHA or was non-agglutinating. The agglutinin(s) of V. anguillarum, however, showed different activity to human, poultry, guinea pig, and rainbow trout erythrocytes and to yeast cells. Therefore, it was found appropriate to design a system based on the agglutination pattern. Similar typing systems have recently been developed for E. coli and Aeromonas hydrophila (1, 13, 19). Although yeast cells seem to be the most sensitive indicator of MSHA, the specificity to the erythrocytes was so pronounced as to warrant the present typing system. Several passages through broth cultures showed that the hemaggluti-

4 1264 LARSEN AND MELLERGAARD nating capacity in most cases persisted but might have been lost to one or more specific species of erythrocytes. Thus, agglutination typing on freshly isolated strains is recommended to accomplish this method. Compared with E. coli it should be expected that an MSHA strain gave a more uniform agglutinating activity (12), but as stated by others (7), a wide variety of chemically diverse adhesins are available to participate in specific cellular associations. However, information on hemagglutinins among V. anguillarum strains is rather sparse. Generally, types A to C showed a higher AT towards erythrocytes than did the other types, whereas no significant difference in the yeast cell AT was observed among types A to E. Therefore, the typing cannot entirely be due to differences in the total production of agglutinin by single strains since it is the lack of agglutinating activity to specific erythrocytes that separates the different agglutination types, e.g., lack of trout erythrocyte agglutination in type B and human erythrocyte agglutination in type C. Use of highly concentrated bacterial cell suspensions in titration of the agglutinating capacity showed significant differences in the agglutination of the single erythrocyte types (Table 4). It appeared that use of a titer of 32, which corresponds to a bacterial cell concentration of 2.5 x 109, was a suitable level in the standard typing system. When the details of the agglutination test are examined, it seems reasonable to use the titer value instead of the more arbitrary AP value. There were no obvious differences in the distribution of the various agglutination types among the fish pathogenic and environmental strains (Table 3). Despite a relatively low number of V. anguillarum strains from the different fish species, there seems to be a tendency toward broad-spectrum agglutination among eel strains, contrary to trout strains, in which yeast-cell agglutination is predominant. The cod isolates show many different agglutination patterns, which is also characteristic for V. anguillarum strains from invertebrates, seawater, and sediment. It is striking that V. anguillarum strains from rainbow trout show little agglutinating activity to rainbow trout erythrocytes. This fact might actually be important in the progress of infection in which agglutination could be fatal for the bacterium, since an attachment to cell surfaces enhances the phagocytosis, resulting in engulfment and perhaps destruction (4). The MRHA of V. ordalii has also been demonstrated by Trust and co-workers (26) with reference to human erythrocytes. This group of workers also found that V. anguillarum 775 exerted MRHA, whereas our results showed that this strain was negative, which was in accordance with very recently published results (25). Loss of agglutinating capacity might be the explanation of this divergence. MRHA of V. anguillarum to erythrocytes from guinea pigs (25) and from humans and trout (26) has been described. However, repeating tests of our collection and the reference strains could not disclose the presence of MRHA by the present test method. The occurrence of a great diversity of hemagglutination types suggests the existence of a multiplicity of V. anguillarum strains in the marine environment, in which many properties still need to be defined. The typing scheme based on hemagglutination patterns might be used as a supplement to serotyping in epidemiological studies, especially in relation to vibriosis in maricultured fish, in which only a few serotypes are involved. As fish pathogenic vibrios are recruited from the environment, it will be of importance to APPL. ENVIRON. MICROBIOL. trace and to obtain information on the source of infection and the possibilities of pollution-induced proliferation of pathogenic strains in selected areas (22, 23). It remains to be proven that agglutinins of V. anguillarum are of pathogenic importance. If this is the case, the selection of bacterial strains for vaccine production must be made with emphasis on the agglutinating capacities. In E. coli, MSHA is associated with the presence of type 1 fimbriae on the bacteria (12). Similar structures have not been detected on V. anguillarum as yet, but other principles might possibly be involved in MSHA of V. anguillarum. These problems should be clarified during studies now in progress. ACKNOWLEDGMENTS The technical assistance of Maj-Britt H0jgaard and typing of the manuscript by Jane R0ken and Jenny J0rgensen are greatly appreciated. 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