Abstract
Vibrio anguillarum and Vibrio alginolyticus exhibited significant adhesion to and chemotactic abilities towards mucus collected from the skin, gills, and intestine of gilt-head sea bream. Quadratic polynomial models for chemotaxis designed to estimate the influence of temperature demonstrated a differential bacterial chemotaxis depending of the source of the mucus, with the chemotaxis towards intestinal mucus being the least influenced.
Vibriosis is one of the most important bacterial diseases that affect farmed gilt-head sea bream (Sparus aurata L.) in southern Europe, with Vibrio anguillarum and Vibrio alginolyticus being two of the species frequently isolated from diseased fish (2). Bacterial adhesion to fish external surfaces is generally the initial step for colonization, and mucus surfaces of the fish can provide a barrier against infection (1). The chemotactic effect exerted on the bacterial strains by different chemical agents is the main factor for the initial stages of the colonization of mucus cellular surfaces (8).
Given the facts that routes of entry of V. anguillarum and V. alginolyticus into S. aurata have not been elucidated yet and that the culture of gilt-head sea bream is carried out under various conditions of temperature and salinity, the scope of this study was to evaluate the chemotaxis of these microorganisms towards mucus collected from different locations of gilt-head sea bream and to study the influence of temperature and salinity on this process.
Twenty-two Vibrio strains isolated from diseased gilt-head sea bream were used to study the adhesion to and chemotaxis towards skin, gill, and intestinal mucus of this fish species. Bacterial culture and incubation conditions were as described by Bordas et al. (3).
Raw skin, gill, and intestinal mucus was prepared following the techniques described by Bordas et al. (3), Lumsden et al. (7), and Olsson et al. (11), respectively. All mucus preparations were adjusted to 800 to 1,000 μg of protein/ml of seawater or phosphate-buffered saline.
Bactericidal and bacteriostatic activities of the mucus and the abilities of the strains assayed to use mucus as the sole nutrient source were determined as previously described (3). The abilities of the strains to adhere to mucus were assayed following the methodology described by Krovacek et al. (6).
Bacterial chemotaxis was measured by the technique described by Milner and Sellwood (8). Capillary tubes (200 μl) were filled with 50-μl mucus solutions. Chemotaxis was expressed as the ratio (Rche) of the number of bacteria in the mucus capillary to the number in the control capillary. Rche values equal to or higher than 2 were considered significant (9). The effects of temperature and salinity on the chemotaxis towards mucus of pathogenic Vibrio strains were also evaluated. Thus, bacterial cultures were prepared in seawater at different conditions of temperature (15, 22, and 27°C) and salinity (10, 17, and 35‰), following the technique described previously (3).
A multivariate statistical analysis of the chemotactic response was developed according to the response-surface technique (10) with a commercial software program (JMP, statistical visualization software for the Macintosh; SAS Institute Inc.).
In this study, skin, gill, and intestinal mucus from gilt-head sea bream did not show inhibitory effects against the Vibrio strains assayed. In addition, lysozyme activity was not detected in the mucus suspensions. Thus, the absence of bactericidal effect in the mucus and the ability of Vibrio strains to use it as the sole nutrient source could favor the colonization of the mucus surfaces of this fish species by these pathogens.
The numbers of adhered cells per square millimeter of mucus are given in Table 1. Strains showing values lower than 103 cells adhered per mm2 were considered negative for adhesion to mucus. Only the strains of V. alginolyticus and V. anguillarum showed adhesive capability for the mucus collected from the three locations assayed. In general, it was possible to observe that the numbers of bacterial cells that adhered to gill mucus were significantly (P < 0.05) lower than the numbers that adhered to skin and intestinal mucus, except for one strain of V. anguillarum (Table 1). On the other hand, the strains of V. anguillarum were significantly (P < 0.05) more adhesive to the three types of mucus assayed than those of V. alginolyticus or strains DC12R7 and DC12R8, which were less adhesive to intestinal mucus. The other strains showed adhesion to only one type of mucus or were not adhesive (Table 1).
TABLE 1.
Numbers of Vibrio bacteria adhering to skin, gill, and intestinal mucus of gilt-head sea bream
| Strain | No. of adhered cells (102)/mm2 of mucusa from:
|
||
|---|---|---|---|
| Skin | Gills | Intestine | |
| V. alginolyticus | |||
| CAN | 25.4 ± 13.2 | 13.0 ± 2.9 | 71.2 ± 14.3 |
| A035 | 32.1 ± 24.8 | 22.2 ± 8.0 | 60.4 ± 33.4 |
| DP1HE4 | 72.3 ± 23.3 | 25.4 ± 6.4 | 67.1 ± 12.4 |
| V. anguillarum | |||
| DC7R2 | 199.1 ± 59.1 | 100.2 ± 13.1 | 140.8 ± 51.6 |
| DC11R2 | 117.1 ± 45.9 | 80.6 ± 12.2 | 129.0 ± 51.1 |
| DC12R7 | 21.8 ± 16.3 | 39.3 ± 14.2 | 12.5 ± 6.8 |
| DC12R8 | 165.7 ± 25.9 | 38.9 ± 7.5 | 21.4 ± 9.1 |
| DC12R9 | 186.1 ± 32.9 | 28.8 ± 7.5 | 191.3 ± 63.7 |
| V. fischeri | |||
| P403 | 11.8 ± 3.8 | 0 | 5.3 ± 3.6 |
| P801 | 14.5 ± 9.6 | 0 | 0 |
| P802 | 0 | 0 | 0 |
| V. harveyi | |||
| DP1U1 | 0 | 0 | 0 |
| DP1U2 | 0 | 0 | 0 |
| DP1HE1 | 0 | 0 | 0 |
| DP1HE2 | 0 | 0 | 45.9 ± 12.6 |
| DP2HE6 | 0 | 0 | 0 |
| V. tubiashii | |||
| DC10R4 | 0 | 0 | 0 |
| Vibrio spp. | |||
| P3H1 | 0 | 0 | 0 |
| P8H3 | 6.8 ± 3.7 | 0 | 0 |
| A028 | 0 | 0 | 0 |
| P4R1 | 0 | 0 | 0 |
| DC10R3 | 0 | 0 | 0 |
The values are arithmetic means of results from 10 experiments.
Ten strains were selected to study the chemotactic process (Table 2). All of them showed significant chemotaxis to all mucus, except the strain of Vibrio fischeri. It was also possible to detect significant (P < 0.05) differences among the strains of V. anguillarum and V. alginolyticus, the former showing a higher level of chemotaxis to all types of mucus than the latter. Strains such as Vibrio harveyi DP1U2 and Vibrio tubiashii DC10R4, which did not show ability to adhere to any type of mucus, exhibited chemotaxis towards all types of mucus. These results suggest that there is not a direct relationship between the chemotactic response to mucus and adhesive ability. Thus, for these strains chemotaxis could play an important role favoring the access to mucus as a nutrient source.
TABLE 2.
Chemotaxis of different Vibrio strains to skin, gill, and intestinal mucus of gilt-head sea bream
| Strain | Background motility | Chemotaxisa towards mucus from:
|
||
|---|---|---|---|---|
| Skin | Gill | Intestine | ||
| V. alginolyticus | ||||
| CAN | 8 × 104 | 13 | 18 | 7 |
| DP1HE4 | 1.5 × 104 | 86 | 73 | 51 |
| V. anguillarum | ||||
| DC7R2 | 1.3 × 103 | 1,030 | 470 | 583 |
| DC11R2 | 4 × 104 | 57 | 178 | 35 |
| DC12R8 | 1.3 × 103 | 958 | 732 | 111 |
| DC12R9 | 1.3 × 103 | 2,708 | 2,629 | 2,297 |
| V. fischeri | ||||
| P403 | 1.3 × 103 | 70 | 24.3 | 0.3b |
| V. harveyi | ||||
| DP1U2 | 1.3 × 103 | 2,455 | 815 | 184 |
| DP1HE2 | 1.3 × 103 | 1,322 | 507 | 45 |
| V. tubiashii | ||||
| DC10R4 | 1.3 × 103 | 246 | 242 | 140 |
Expressed as Rche values.
Nonsignificant Rche value.
V. alginolyticus CAN and V. anguillarum DC12R9 were selected to evaluate the effect of temperature and salinity on the chemotaxis towards mucus. V. alginolyticus CAN showed minimal chemotaxis towards skin and gill mucus at extreme values of salinity and at temperatures of 15°C for skin mucus and 27°C for gill and intestinal mucus (Table 3). The DC12R9 strain showed a minimal chemotactic response towards mucus with high salinity values and at 22°C for skin and intestinal mucus and 15°C for gill mucus. The Rche values obtained in these experiments in which the strains were preincubated for 12 h in seawater, a nutrient-deficient medium, are lower than those obtained when the strains assayed were grown in tryptic soy broth (Difco Laboratories, Detroit, Mich.) with 1.5% NaCl and then resuspended in phosphate-buffered saline.
TABLE 3.
Influence of temperature and salinity on the chemotaxis of V. anguillarum and V. alginolyticus towards skin, gill, and intestinal mucus of gilt-head sea bream
| Strain and mucus type |
Rchea at indicated temp and salinity
|
||||||||
|---|---|---|---|---|---|---|---|---|---|
| 15°C
|
22°C
|
27°C
|
|||||||
| 10‰ | 17‰ | 35‰ | 10‰ | 17‰ | 35‰ | 10‰ | 17‰ | 35‰ | |
| V. anguillarum DC12R9 | |||||||||
| Skin | 25.9 | 13.3 | 18.5 | 20.9 | 130.1 | 3.8 | 121.8 | 101.1 | 17.9 |
| Gill | 26.11 | 11.0 | 7.4 | 27.9 | 10.6 | 10.4 | 73.6 | 81.5 | 37.4 |
| Intestine | 14.07 | 17.5 | 9.3 | 4.8 | 20.4 | 3.0 | 4.0 | 58.9 | 11.9 |
| V. alginolyticus CAN | |||||||||
| Skin | 9.3 | 4.5 | 6.3 | 53.4 | 33.4 | 8.2 | 53.8 | 128.9 | 13.4 |
| Gill | 3.5 | 4.1 | 6.2 | 16.3 | 15.4 | 11.7 | 35.9 | 64.2 | <2b |
| Intestine | 10.0 | 6.8 | 7.7 | 21.8 | 8.1 | 6.8 | 4.0 | 5.2 | <2b |
The values are arithmetic means of results from three experiments.
Nonsignificant Rche value.
The results obtained demonstrated that the assayed variables exerted a variable influence on the chemotaxis depending on the mucus location. By response surface analysis, it was possible to generate quadratic polynomial models for chemotaxis (Q) in terms of temperature (T) and salinity (S), represented by equations of the form Q = Q0 + aT + bS + cT2 + dST + eS2, for each strain and type of mucus. The r2 values for chemotaxis models were >0.89. These models provide an estimation of the chemotaxis process in response to combinations of variables studied within the specified ranges. Thus, it was observed that chemotaxis to skin mucus is more influenced by the temperature and salinity than chemotaxis to gill or intestinal mucus, the latter being the least affected. The models were used to predict nonsignificant chemotaxis for additional experimental conditions. The experimental results in these predicted conditions validated the model’s predictive ability for chemotaxis to S. aurata mucus.
The conditions of temperature and salinity in which the culture of gilt-head sea bream is carried out ranged between 25 and 40‰ and 18 and 25°C. These ranges were applied to the model, and the results obtained show that the chemotaxis of strain CAN towards intestinal mucus in the ranges studied would be significant in practically all cases, while chemotaxis to gill and skin mucus would not be significant in some conditions. On the other hand, strain DC12R9 may have significantly higher Rches for intestinal and skin mucus than for gill mucus.
The results obtained from the adhesion and chemotaxis experiments suggest that the mucus surfaces are potential portal entries in the fish for V. anguillarum and V. alginolyticus. It could be assumed that the intestine is a more probable colonization area than skin and gills in the environmental conditions of temperature and salinity of farmed gilt-head sea bream. This is in accordance with data reported by several authors, who suggested that the intestine is an important portal entry for V. anguillarum (11) and that intestinal mucus is a good medium from which to recover starved cells of this microorganism (4). We have previously demonstrated that the infection of larval gilt-head sea bream by Vibrio species, including V. alginolyticus, via the food chain could lead to high mortality (12), and, in addition, Grisez et al. (5) demonstrated that V. anguillarum was transported through the intestinal epithelium of turbot larvae. These results support the idea that the intestinal via may be an important portal entry for V. alginolyticus and V. anguillarum and that chemotaxis towards intestinal mucus could be responsible for the detachment of the bacteria from the surfaces of the zooplankton and the movement towards the mucus.
Acknowledgments
This work was funded by a grant of the Comisión Interministerial de Ciencia y Tecnología (C.I.C.Y.T.) of the Spanish Government (MAR97-1188-CO2-O2).
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