Vibrio salmonicida The bacterium

Vibrio salmonicida, the agent of Hitra disease, a cold water vibriosis affecting Atlantic salmon (Salmo salar), is a facultatively anaerobic motile rod. It possesses several polar flagella (Fig. 14.8) (Egidius et al, 1986), and, when isolated from fish, it shows a high degree of pleomorphism. Vibrio salmonicida is a halophilic bacterium that grows in the presence of 0.5-4% NaCl with an optimum salt concentration of 1.5% (Egidius et al, 1981). The bacterium is

Vibrio Parahaemolyticus
Fig. 14.8. Electron micrograph of Vibrio salmonicida showing a polar tuft of nine sheathed flagella. x 7120. Bar = 1 mm. (From Egidius et al., 1986, with permission from the authors and the American Society for Microbiology.)

psychrophilic and can grow between 1°C and 22°C, with an optimum temperature of 12-15°C. Colonies in nutrient agar supplemented with blood are small and greyish, showing no pigmentation (Egidius et al, 1986). Vibrio salmonicida is non-haemolytic when grown on solid media containing either human or sheep blood. This pathogen could be isolated from sediments obtained from farms in which fish had had V. salmonicida vibriosis but were disease-free up to 7 months prior to the study (Hoff, 1989), and is also isolated from marine sediments from disease-free farms. However, this bacterium was not detected in sediments of regions not influenced by fish farming. Since V. salmonicida has been isolated from faeces of experimentally infected fish, these results suggested that V. salmonicida could reach the marine sediments through the fish faeces and remain there for long periods of time (Enger et al, 1989). In addition, healthy fish can be carriers and they contribute to its spread in the marine environment.

Vibrio salmonicida is serologically distinct from V. anguillarum. No immunological cross-reaction was detected with three strains of V. anguillarum, using rabbit polyclonal antibodies raised against V. salmonicida (Egidius et al, 1986). However, Enger et al. (1989) showed that polyclonal antibodies against V. salmonicida can cross-react with V. anguillarum 775 and V. ordalii PT1 strains isolated from cod (Gadus morhua), as well as against uncharacterized strains isolated from marine sediments. Conversely, the utilization of MAbs allowed the detection of V. salmonicida only (Enger et al., 1989).

Vibrio salmonicida strains are differentiated by their plasmid profiles (S0rum et al, 1988, 1990; Wiik et al, 1989a,b). Common plasmid-isolation techniques such as the alkaline extraction procedure described by Birnboim and Doly (1979) or that described by Kado and Liu (1981), gave poor results, however, when applied to V. salmonicida (Wiik et al, 1989a,b). An alternative method which did not include the alkaline denaturation step proved to be adequate for plasmid isolation from this bacterium (Orberg and Sandine, 1984; Wiik et al, 1989a,b). Plasmids of 170, 61, 24, 10, 3.4 and 2.8 MDa were detected in V. salmonicida isolated from diseased cod and Atlantic salmon (S0rum et al, 1988; Wiik et al, 1989a,b). Certain plasmid profiles were characteristic of strains isolated in different geographical regions on the Norwegian coast (Table 14.3). This table shows that the 61 MDa plasmid is only present in strains isolated from northern Norway, while the 21 MDa plasmid seems to be ubiquitous. The relationship among these plasmids was studied by DNA-DNA hybridization. The only homology found was between the 24 and the 2.8 MDa plasmids.

Epidemiological studies based on plasmid profiles suggest that V. salmonicida was transmitted from cod to Atlantic salmon and vice versa in fish farms in northern Norway (S0rum et al, 1990). No correlation could be found between the plasmid profiles and the LD50 values of the strains analysed. Valla et al. (1992) has analysed the 10 MDa plasmid pVS1 present in the strain TE083.00. This study shows that this plasmid harbours repeated sequences as well as sequences homologous to other plasmids present in other isolates of V. salmonicida. A curing method for pVS1 was developed based on plasmid incompatibility, since the utilization of chemical agents was ineffective. A recombinant derivative of the IncQ plasmid pPV14 harbouring pVS1 incompatibility determinants (pPV14.16) was introduced by conjugation into the V. salmonicida TEO83.001 strain. Selection after the conjugation for the incoming pPV14.16 plasmid resulted in the loss of pVS1. Further growth of this exconjugant strain in the absence of selective pressure resulted in a plasmid-free derivative, due to the instability of pPV14.16 in V. salmonicida TE083.001. Experimental infections and serotype analysis of the wild type and the cured

Table 14.3. Plasmid patterns of Vibrio salmonicida strains isolated from different regions of the Norwegian coast (data from S0rum et al., 1990).

Plasmid pattern (MDa)

Geographical region of Norwegian coast*

Northern

Central

Western

61, 21, 3.4, 2.8

14

61, 21

1

21, 3.4. 2.8

20

1

3

21, 3.4

13

38

24

21

3

6

*Number of V. salmonicida strains isolated along the Norwegian coast containing plasmids of the indicated sizes.

*Number of V. salmonicida strains isolated along the Norwegian coast containing plasmids of the indicated sizes.

derivative demonstrated that there is not a significant correlation between the plasmid content and the virulence of V. salmonicida TE083.001. Furthermore, the curing of this plasmid did not affect the metabolic properties of this strain.

The largest plasmid of 170 MDa has recently been characterized and found to carry a tetracycline resistance gene (S0rum et al, 1992). In Norway, oxytetracycline is the current treatment against V. salmonicida, and during the last 6 years no tetracycline resistant V. salmonicida has been detected. However, lately several resistant strains were isolated (S0rum et al., 1992). For one strain, the tetracycline resistance gene was identified and cloned from the 170 MDa plasmid, named pRSV1. By hybridization analysis it was found that it has homology to the tetA(E) gene isolated from a human E. coli isolate and strains of Aeromonas hydrophila (S0rum et al., 1992). The expression of this gene, encoding a 26.5 kDa protein, appears to be regulated by the concentration of tetracycline in the growth medium. Removal of a DNA fragment rendered expression of this gene constitutive, suggesting the presence of a regulatory system, as was already described for the E. coli tetA(E) gene (Tovar et al, 1988). Recently, Andersen and Sandaa (1994) showed, using DNA probes for the tetracycline resistance determinants A to E, that E is the most dominating determinant detected in polluted and unpolluted marine sediments isolated from Norway and Denmark. These investigators suggested that the altered distribution of antibiotic resistance observed during this study may be the consequence of the increased consumption of tetracycline for human and veterinary purposes, the disposal of faecally contaminated water in the marine environment, along with the appearance and spread of antibiotic-resistant microorganisms.

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