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CAB INTERNATIONAL 1999. Fish Diseases and Disorders, Volume 3:

Viral, Bacterial and Fungal Infections (eds P.T.K. Woo and D.W. Bruno)
14
Vibriosis
L.A. Actis,
1
M.E. Tolmasky
2
and J.H. Crosa
3*
1
Department of Microbiology, Miami University, Oxford, Ohio 45056, USA;
2
Institute of Molecular Biology and Nutrition, Department of Biological
Science, School of Natural Sciences and Mathematics, California State
University Fullerton, Fullerton, California 92834-6850, USA;
3
Department of
Molecular Microbiology and Immunology, School of Medicine, Oregon Health
Sciences University, Portland, Oregon 97201-3098, USA.
INTRODUCTION
Vibriosis is one of the most prevalent fish diseases caused by bacteria belonging
to the genus Vibrio. Vibriosis caused by Vibrio anguillarum has been particularly
devastating in the marine culture of salmonid fish. The causative agent,
V. anguillarum, was first described in 1909 by Bergman as the aetiological agent
of the red pest of eels in the Baltic Sea. Before this report, Canestrini (1893)
described epizootics in migrating eels (Anguilla vulgaris) dating back to 1817
that implicated a bacterium named Bacillus anguillarum. The pathology of the
disease and the characteristics of the bacterium in these two reports suggested
that the etiological agents were the same. Vibriosis was not reported in North
America until 1953, when V. anguillarum was isolated from chum salmon
(Oncorhynchus keta) (Rucker et al., 1953). Outbreaks affecting close to 50
species of fresh- and salt-water fishes have been reported in several countries in
the Pacific, as well as the Atlantic coasts (Anderson and Conroy, 1970; Strout et
al., 1978; Tolmasky et al., 1985; 1988a). The losses produced by this disease are
so disastrous that vibriosis caused by V. anguillarum has been recognized as a
major obstacle for salmonid marine culture (Schiewe, 1983; Winton et al., 1983;
Trust, 1986). Several years ago, Harrell et al. (1976) demonstrated that isolates
of V. anguillarum, the most important aetiological agent of vibriosis, exhibited a
marked heterogeneity, which led to the division of these vibrios into two
separate biotypes, 1 and 2 (Schiewe et al., 1977). Later on, Schiewe et al. (1981)
proposed a new species for the V. anguillarum biotype 2, based on cultural and
biochemical characteristics, as well as in deoxyribonucleic acid (DNA)
homology with the biotype 1. This new species was named Vibrio ordalii in
honour of Erling J. Ordal.
Other members of the genus Vibrio have been isolated in outbreaks of
vibriosis in fish and shellfish. These aetiological agents include Vibrio
*Corresponding author.
524
L.A. Actis et al.
salmonicida, a pathogen isolated on the Norwegian coast, causing Hitra disease
or cold water vibriosis; Vibrio damsela (Love et al., 1981); Vibrio vulnificus
biotype II (Tison et al., 1982); Vibrio tubiashii (Hada et al., 1984); Vibrio
carchariae (Grimes et al., 1984); and Vibrio cholerae non-O1 (Muroga et al.,
1979; Yamanoi et al., 1980). Vibrio splendidus and Vibrio pelagius, atypical V.
anguillarum strains and strains from the environment were recently isolated
from infected fish on the Atlantic coast of Spain and Norway (Toranzo and
Barja, 1990).
AETIOLOGICAL AGENTS OF VIBRIOSIS
Vibrio anguillarum
The bacterium
Vibrio anguillarum is a polarly flagellated, Gram-negative, curved rod (Fig.
14.1). It is a facultative anaerobe, with a guanine plus cytosine (G + C) content of
4346%. It grows rapidly at 2530C in rich media, such as brain-heart,
trypticase soy broth or agar containing 1.5% sodium chloride (NaCl). On solid
medium, it produces circular, cream-coloured colonies. Vibrio anguillarum
belongs to one of the halophilic groups of vibrios and survives at different
salinities. Hoff (1989) has shown that it is able to survive in sea water for more
than 50 months. A list of the phenotypic properties, as well as the salt and
temperature range for V. anguillarum, has been published (Schiewe et al., 1981).
In the recent past, new names have been proposed for V. anguillarum, such
Fig. 14.1. Electron micrograph of V. anguillarum 775 showing single polar flagellum. Shadowed
preparation 10,000. (Micrograph by Dr J .H. Crosa.)
525 Vibriosis
as Beneckea anguillara biotype 1 (Baumann et al., 1978) or Listonella
anguillarum (McDowell and Colwell, 1985). For historical and practical reasons
and because of its close relationship to other marine vibrios (Crosa, 1989;
Tolmasky et al., 1994; Wertheimer et al., 1994), we shall continue to use the
original nomenclature, i.e. V. anguillarum, for this important fish pathogen.
The disease
Vibriosis occurs in cultured and wild marine fish in salt or brackish water,
particularly in shallow waters during late summer. It was originally believed that
scavenger fish feeding around the farms were the natural reservoir of
V. anguillarum, and contact between fish seems to be an important factor for the
spread of this pathogen. However, there is evidence that V. anguillarum is
normally present in the food of cultured and wild healthy fish (Roberts, 1989).
The temperature and quality of the water, the virulence of the V. anguillarum
strain and stress on the fish are important elements influencing the onset of
disease outbreaks. The characteristic clinical signs of vibriosis include red spots
on the ventral and lateral areas of the fish and swollen and dark skin lesions that
ulcerate, releasing a blood exudate (Fig. 14.2). There are also corneal lesions,
characterized by an initial opacity, followed by ulceration and evulsion of the
orbital contents. However, in acute and severe epizootics, the course of the
infection is rapid, and most of the infected fish die without showing any clinical
signs. A recent review (Toranzo and Barja, 1990) details all reports of
V. anguillarum-caused vibriosis in cultured fish, molluscs and crustacea. Table
14.1, adapted from this publication, lists the vibriosis outbreaks caused by
V. anguillarum in different countries.
Recently, Ransom et al. (1984) studied and compared the histopathology of
vibriosis caused by V. anguillarum and V. ordalii in rainbow trout and salmon
and found significant differences between the forms of disease caused by these
bacteria. In this section, we shall describe vibriosis caused by V. anguillarum.
This disease is characterized by a haemorrhagic septicaemia. The number of
leucocytes is reduced (Ransom et al. 1984), and darkening of the diseased fish is
Fig. 14.2. J uvenile coho salmon (Oncorhynchus kisutch) exhibiting some of the common signs
of vibriosis. The extensive haemorrhaging along the lateral line and abdominal surfaces is typical
of the chronic form of this disease. (Photograph obtained from Michael Schiewe, National Marine
Fisheries Service.)
526
L.A. Actis et al.
Table 14.1. Vibriosis outbreaks caused by Vibrio anguillarum (from Toranzo
and Barja, 1990).
Species Country
Pacific salmon
Oncorhynchus kisutch USA, J apan, Spain
O. keta, O. nerka, O. gorbuscha Canada
O. masou, O. rhodurus J apan
O. tshawytscha USA, Canada
Atlantic salmon
Salmo salar Norway
Trout
Oncorhynchus mykiss USA, J apan, Italy, Norway,
Denmark, Spain
Salmo trutta Scotland
Turbot
Scophthalmus maximus Scotland, Spain
Striped bass
Morone saxatilis USA
Winter flounder
Pseudopleuronectes americanus USA
Cod
Gadus morhua Norway, Denmark
Red sea-bream
Pagrus major J apan
European eel
Anguilla anguilla Norway
J apanese eel
Anguilla japonica J apan
Saithe
Pollachius virens Norway
Gilthead sea-bream
Sparus aurata Israel
Sea mullet
Mugil cephalus Scotland
Seriola
Seriola quinqueradiata J apan
Channel catfish
Ictalurus punctatus USA
Milkfish
Chanos chanos Taiwan
Ayu
Plecoglossus altivelis J apan
Tilapia
Oreochromis aureus Kuwait
European oyster
Ostrea edulis USA, UK, Spain
J apanese oyster
Crassostrea virginica USA
Clam
Mercenaria mercenaria USA
Lobster
Homarus americanus USA
Shrimp
Penaeus sp. USA
527 Vibriosis
noted, with petechiae at the base of fins and skin. Ulcers can also be observed
(Fig 14.2). Cisar and Fryer (1969) reported that the intestine becomes distended
and fills with a clear, viscous liquid. Vibrio anguillarum was found in large
numbers in the blood and haemopoietic tissues. The pathology is more severe in
the descending gastrointestinal tract and rectum than in the anterior region, due
to a pH gradient, which is alkaline in the rectum and becomes acidic towards
the anterior gastrointestinal tract. Ransom et al. (1984) demonstrated that
V. anguillarum cannot grow in an acidic medium. Histological examination of
infected rainbow trout tissues can demonstrate the location of the V. anguillarum
during infection (Nelson et al., 1985a,b). The bacterium was initially found in
the spleen, but, as the number of cells in this organ increased, bacteria appeared
in the kidney. At the time of death, most tissues were septic and no phagocytosis
by macrophages was detected (Nelson et al., 1985b). Severe cardiac myopathy,
renal and splenic necrosis and periorbital oedema were also described in
preacute vibriosis cases (Roberts, 1989). A comparison of pathological changes
caused by V. anguillarum and its extracellular products in rainbow trout
(Oncorhynchus mykiss), as well as an analysis of the non-specific cellular
responses of rainbow trout to these products, has been recently described
(Lamas et al., 1994a,b).
Identification and classification
Outbreaks of V. anguillarum may lead to a rapid loss of farmed fish. Therefore, a
quick diagnosis to minimize fish losses is essential. Identification methods
include a culture medium for presumptive identification, a sensitivity assay to
filter discs impregnated with a saturated solution of the vibriostatic agent 0/129
(2,4-diamino-6,7-diisopropylteridine), nitrate reduction, presence of oxidase,
catalase and arginine decarboxylase, reaction with monoclonal antibodies and
antiflagellar antiserum, and hybridization with specific 16S ribosomal
ribonucleic acid (rRNA) oligonucleotides (Shewan et al., 1954; Larsen, 1983;
Tassin et al., 1983; Rehnstam et al., 1989; Alsina et al., 1994; Martinez-Picado
et al., 1994). Identification of Vibrio species that possess similar biochemical
and morphological properties can be achieved by using monoclonal antibodies
(MAbs) prepared against sodium azide-killed cells (Chen et al., 1992). Three
types of MAbs were analysed and enzyme-linked immunosorbent assays
(ELISA) and fluorescein isothiocyanate (FITC) immunofluorescence tests
showed that the genus-specific MAbs were very useful for identifying vibrios,
while the species-specific MAbs were useful for completing the diagnosis. Most
of these biochemical and immunological methods require the culture and
isolation of the infecting bacteria from the fish. Conversely, newer hybridization
techniques do not require a pure culture. Rehnstam et al. (1989) and Martinez-
Picado et al. (1994) described synthetic oligonucleotides used as specific probes
for the identification of V. anguillarum. These oligonucleotides were designed
using the information generated after sequencing the 16S rRNA from several
V. anguillarum strains. The radiolabelled nucleotides were used as probes in
DNA hybridization assays, which are carried out using purified DNA and
homogenized fish tissues, such as kidney. The hybridization of these probes
against V. anguillarum DNA is very specific, with no cross-hybridization against
528
L.A. Actis et al.
other bacterial species. This molecular assay permits the identification of
V. anguillarum within 24 h.
In comparative studies, pathogenic, environmental and reference strains are
similar, but no cross-reacting antigens are present in these strains (Larsen, 1983).
Therefore, the classification of V. anguillarum by serological methods has
proved to be convenient (Pacha and Kiehn, 1969; Johnsen, 1977; Kitao et al.,
1983; Srensen and Larsen, 1986). A serotyping scheme has been proposed
based on the detection by slide agglutination of V. anguillarum O antigens
(Srensen and Larsen, 1986). Using this test, Toranzo et al. (1987) detected the
presence of this pathogen in infected fish. Ten serotypes (O1O10) have been
described in V. anguillarum; however, most vibriosis outbreaks involving
cultured salmonid fish and feral marine fish have been shown to be caused by
strains belonging to serotypes O1 and O2, respectively (Toranzo and Barja,
1990). Strains belonging to serotypes O3 to O10 have been mainly isolated from
marine environmental samples, including water, sediment and phyto- and
zooplankton. Recently, Bolinches et al. (1990) used a combination of
immunological methods, including double immunodiffusion, dot-blot assay and
an enzyme-linked immunoabsorbent technique, and established that the O2
group can be subdivided into two subgroups, O2 and O2. This finding agreed
with that of Rasmussen (1987b), who demonstrated that two lipopolysaccharide
species, nominated O2a and O2b, were present in O2 strains. Bacteria belonging
to the O2a group were isolated from salmonids and non-salmonid fish, while
strains belonging to the O2b group were isolated only from non-salmonids. The
question of whether the heterogeneity observed within the O2 also occurs in
other serotypes awaits further study.
The presence of capsular antigens (K antigens) has been reported for
V. anguillarum of the O1, O4, O5 and O6 groups (Rasmussen, 1987a,c; Tajima et
al., 1987a,b). However, the role of K antigens in pathogenicity remains to be
determined.
The correlation between serotype and pathogenicity may reflect the ability
of the bacterial surface antigens to interact with the hosts tissues. Furthermore,
studies of surface components, such as outer-membrane proteins and lipopoly-
saccharides, of V. anguillarum strains demonstrated that these bacterial
components are related to the serotypes of the pathogens (Aoki et al., 1981;
Nomura and Aoki, 1985).
Agglutination has also been used to type environmental and fish isolates
(Larsen and Mellegaard, 1984). All V. anguillarum isolates either exerted
mannose-sensitive haemagglutination or were non-agglutinating. The
V. anguillarum agglutinins have specificity against human, poultry, guinea pig
and trout erythrocytes, as well as yeast cells. Therefore, a scheme was developed
based on the agglutination ability of the different isolates for specific eukaryotic
cells. Eight different agglutination types (AH) were defined by this method.
Treatment and prophylaxis
Control of V. anguillarum, as well as other vibrios, such as V. ordalii, includes
the use of antibiotics, vaccination and the culture of other bacteria that inhibit the
growth of V. anguillarum. Antibiotics and other chemotherapeutic agents are
529 Vibriosis
used as feed additives or added directly to the water to prevent and treat
vibriosis. In Japan, ampicillin, chloramphenicol, nalidixic acid derivatives,
nitrofuran derivatives, sulphonamides and trimethoprim have been routinely
used to treat vibriosis (Aoki et al., 1984). However, the use of these compounds
has resulted in drug resistant strains (Watanabe et al., 1971; Shotts et al., 1976;
Aoki et al., 1977, 1979, 1980; Hayashi et al., 1982; Toranzo et al., 1984).
Tetracycline resistant isolates were recovered from cultured ayu (Plecoglossus
altivelis), up until 1977, when the use of this antibiotic was discontinued.
Analysis of the resistance profile of V. anguillarum recovered since 1978 has
shown that only one isolate was resistant to tetracycline (Aoki et al., 1984),
demonstrating a correlation between the use of tetracycline and the appearance
of resistance. Molecular and genetic analysis show that the genes encoding
antibiotic resistance were often found in plasmids. Furthermore, in some cases
these plasmids were shown to be conjugative (Aoki et al., 1984; Toranzo et al.,
1984). Further studies are needed to demonstrate whether these genes are also
present in transposable elements (Kleckner, 1981; Bennet and Hawkey, 1991) or
integrons (Stokes and Hall, 1989).
Traditional vaccines consist of formalin-killed V. anguillarum or bacterial
membrane components (Agius et al., 1983) and give the best protection when
administered by intraperitoneal injection rather than by immersion or oral
administration (Kawano et al., 1984; Ward et al., 1985), but the former method
has limitations and, for practical reasons the other approaches are currently used.
Recent attempts to develop a live vaccine, using avirulent mutants, have used
transposition mutagenesis. Norqvist et al. (1989) constructed avirulent mutants
capable of inducing protective immunity after bath vaccination. Avirulent strains
were constructed by curing of the virulence plasmid pJM1(Crosa et al., 1980),
by performing other genetic manipulations on this plasmid, such as deletion
of the iron-uptake region (Walter et al., 1983), and mutagenesis by either
transposition (Tolmasky et al., 1988a) or in vitro insertion (Singer et al., 1991).
In this latter case, the avirulent derivative persisted in inoculated fish. The
mutants could be isolated 9 days postinoculation, suggesting that these avirulent
V. anguillarum derivatives could be good candidates for a live attenuated
vaccine.
Another strategy to combat vibriosis has been recently proposed, based on
observations that bacteria of the normal gut flora produce inhibitory substances
(Lemos et al., 1985; Onarheim and Raa, 1990; Westerdahl et al., 1991). After
studying more than 400 intestinal isolates from turbot (Scophthalmus maximus,
L.), Westerdahl et al. (1991) found that 28% of those isolates exhibited
inhibitory effects against V. anguillarum. Thus, it is at least theoretically possible
that strains with enhanced inhibitory action could provide protection after oral
administration and might be generated by genetic and molecular biology
methods.
Virulence factors
Several virulence factors have been identified. One virulence factor of the
prototype O1 strain V. anguillarum 775 is a very efficient iron-sequestering
system, encoded by the 65-kilobase pair (kb) pJM1 plasmid (Crosa et al., 1977;
530
L.A. Actis et al.
for recent reviews, see Crosa, 1984, 1987, 1989; Tolmasky and Crosa, 1990;
1991). The components of the system are a siderophore, named anguibactin, and
a membrane-receptor complex. Figure 14.3 shows schematically how this
system functions. Anguibactin is excreted outside the cell and competes for iron
with the high-affinity iron-binding proteins present in the fish. The Fe(III)
anguibactin complex is specifically recognized by the membrane receptor and
internalized into the cell. The biosynthesis of the components of this iron-uptake
system, i.e. the extracellular siderophore anguibactin and the membrane
transport complex, is induced when bacteria are growing under iron-limiting
conditions. Curing of pJM1, as well as Tn1 insertions in the pJM1 iron-uptake
region are correlated with a significant reduction in the ability of V. anguillarum
Fig. 14.3. Model of the pJ M1-mediated iron uptake system. The pJ M1 iron uptake region
(I.U.R.), containing structural and regulatory genes for enzymes involved in the biosynthesis of
anguibactin and proteins of the receptor complex, is shown. The genetic region encoding the
regulator trans-acting factor (TAF) is also indicated. The receptor complex is shown enlarged, and
the putative proteins involved in the transport process are schematically depicted in their location
in the membranes or periplasmic space.
531 Vibriosis
to obtain iron from the culture medium, and a concomitant impairment in
virulence (Crosa, 1980; Crosa et al., 1980, 1983, 1985; Walter et al., 1983). The
role of this iron-uptake system in virulence was further confirmed by the
introduction of recombinant clones possessing the pJM1-encoded iron-uptake
genetic determinants into avirulent V. anguillarum strains, in which a gene or
genes encoding functions for the iron-uptake system were mutagenized, either
by insertion or by deletion. The transconjugant strains recovered the ability to
take up iron efficiently and became virulent (Tolmasky and Crosa, 1984). The in
vivo expression of the pJM1-encoded iron-uptake system was determined by
carrying out experimental coinfections, using the 775 wild-type strain and an
avirulent insertional mutant, affected in the anguibactin biosynthesis but not in
the receptor activity for this siderophore (Wolf and Crosa, 1986). These
experiments demonstrated that the mutant impaired in the synthesis of
anguibactin could be recovered from dead fish only when coinjected with the
775 wild-type strain. Therefore, Wolf and Crosa (1986) demonstrated that
anguibactin is indeed produced and secreted into the bloodstream of the natural
host during the process of infection. Once in the bloodstream, anguibactin can
scavenge iron from the host and deliver this essential metal to the bacterial
cytosol. These experiments showed not only that anguibactin is produced in vivo
but also that it is an important factor of bacterial virulence.
The siderophore anguibactin has been purified from the supernatant in
V. anguillarum 775 cultures grown under iron starvation (Actis et al., 1986) and
characterized as -N-hydroxy--N((2-(2,3-dihydroxyphenyl)thiazolin-4-yl)
carboxy)histamine (Fig. 14.4) (Jalal et al., 1989). Although most siderophores
can be classified into two main groups catechols and hydroxamates (Neilands,
1983) anguibactin belongs to a unique structural class of its own, harbouring a
catechol moiety together with a hydroxamate residue. The stoichiometry of
anguibactin to ligand was determined in vitro, using Ga(III), and it was shown to
be 1:1 (Jalal et al., 1989).
Recently, it was reported that strains of the genus Acinetobacter secrete
acinetobactin, a high-affinity siderophore related to anguibactin (Yamamoto
et al., 1994). Structure analysis showed that acinetobactin is an iron chelator,
containing an amino acid linked to dihydroxybenzoic acid and hydro-
xyhistamine. The only difference between these two iron-scavenging com-
pounds is that anguibactin contains cysteine, while threonine is the amino acid
found in acinetobactin as a functional group. Further analysis, using siderophore
utilization bioassays, showed that acinetobactin enhances the growth of V.
anguillarum under iron-stress conditions (L.A. Actis and J.H. Crosa,
unpublished results).
All these results showed, for the first time, that the V. anguillarum
anguibactin-mediated iron-acquisition system has a homologue in a different
bacterial genus. However, the genes encoding these systems have no detectable
homologies, suggesting that their nucleotide sequences must have diverged after
their transfer by a mechanism that remains to be determined. Transformation,
conjugation and transposition occur in Acinetobacter (Vivian, 1991) and the
pJM1 iron-uptake genes are flanked by insertion sequences in a composite
transposon-like structure (Tolmasky and Crosa, 1995). Furthermore,
532
L.A. Actis et al.
Acinetobacter is a component of the bacterial flora of salmonid fishes (Cahill,
1990). Thus, plasmid conjugation, transformation and/or transposition might
have played a role in the transmission of these essential genes between these two
unrelated bacterial strains present in the microbial flora of salmon and trout.
The membrane transport components involved in the recognition and
internalization of the Fe(III)anguibactin complexes include at least four
Fig. 14.4. Structure of anguibactin. The ferric ion is coordinated by the O-hydroxy group, the
nitrogen of the thiazoline ring, the hydroxamate and the deprotonated nitrogen of the imidazole
ring (J alal et al., 1989).
533 Vibriosis
proteins: FatA, FatB, FatC, and FatD, which were shown to be encoded by the
pJM1 plasmid (Actis et al., 1988; Koster et al., 1991) (Fig. 14.3). Sequence
analysis of the pJM1 region encoding these proteins showed homologies
between these transport components and those described in Escherichia coli for
the transport of Fe(III)-citrate, Fe(III)-hydroxamates and catecholates and
vitamin B
12
(Koster et al., 1991). Of the four components, the best characterized
is the 86-kDa FatA protein, which is only detected when cells are grown under
iron-limiting conditions (Crosa and Hodges, 1981). This protein is located on the
outer membrane and is exposed to the extracellular environment (Actis et al.,
1985). Analysis of the deduced amino acid sequence of FatA demonstrated that
this protein has homologous regions to seven E. coli receptor proteins involved
in the uptake of siderophores and vitamin B
12
(Koster et al., 1991). This finding
supports the idea of a common ancestral gene for these bacterial receptor
proteins. Analysis of the FatA sequence also revealed the presence of a TonB box
in FatA suggesting the presence of an as yet unidentified TonB-like protein in
V. anguillarum. The TonB protein is presumably responsible for energy coupling
to the outer membrane (Heller et al., 1988; Pressler et al., 1988). FatB is the
putative anguibactin binding protein. It is a lipoprotein that is anchored in the
inner membrane facing the periplasmic space (Actis et al., 1995), while FatD
and FatC are integral membrane proteins (Koster et al., 1991). Based on their
homologies to other iron-transport systems, these last two proteins appear to be
involved in the translocation of the Fe(III)-anguibactin complexes across the
cytoplasmic membrane (Koster et al., 1991).
To study the genetics as well as the molecular biology of the pJM1-encoded
Fig. 14.5. Bioassay to detect levels of anguibactin activity. Minimal-medium plates were seeded
with an indicator Vibrio anguillarum strain which has the receptor for Fe(III)-anguibactin but does
not produce the siderophore. Samples of partially purified product from V. anguillarum cultures
were added to the filter disc and placed on top of the agar. The growth of the indicator strain
around the paper disc gives an idea of the amount of anguibactin added. The discs were spotted
with: A, 1 l of product from V. anguillarum harbouring the iron-uptake region clone in addition to
pJ HC-T6.14; B, 10 l of product from V. anguillarum harbouring the iron-uptake region clone
without pJ HC-T6.14. (From Tolmasky et al., 1988a, with permission from the authors and the
American Society for Microbiology.)
534
L.A. Actis et al.
iron-uptake system, the DNA region encoding all the elements of this system
was cloned in the low-copy-number wide-host-range cosmid pVK102 (Knauff
and Nester, 1982). Thus, the recombinant clones generated in E. coli strains
could replicate in avirulent V. anguillarum derivatives upon conjugational
transfer (Tolmasky and Crosa, 1984). One of such clones, pJHC-T2612, was
able to promote the biosynthesis of all components of the receptor complex as
well as anguibactin, upon transfer to the plasmidless avirulent V. anguillarum
H775-3 strain. However, this siderophore was produced in lower amounts when
compared with the 775 wild-type strain. These results suggested that pJM1 also
harboured genetic determinants for a factor necessary for full expression of
genes involved in the biosynthesis of anguibactin. Gene(s) coding for this factor
were cloned, using either pJHC-T00181 (Tolmasky et al., 1986) or pBR325
(Bolivar, 1978) as vectors. Some of the clones generated for example pJHC-
T6.14, which contained a DNA fragment non-contiguous (see Fig. 14.3; trans-
acting factor (TAF) region) to the iron-uptake region (cloned in pJHC-T2612)
were transferred to V. anguillarum already harbouring pJHC-T2612, and the
transconjugant strain was tested for its ability to produce normal levels of
anguibactin. As shown in Fig. 14.5, V. anguillarum harbouring pJHC-T2612
together with pJHC-T6.14 produced higher levels of anguibactin when
compared with those excreted by V. anguillarum harbouring only pJHC-T2612.
Therefore, the existence of a regulator that can act in trans (TAF) encoded by
pJM1 was proved (Tolmasky et al., 1988a). It was later shown that this regulator
acts synergistically with another regulator, AngR, encoded within the iron-
uptake region to promote transcription of genes involved in the iron-uptake
system (Salinas et al., 1989).
The genetic analysis of the iron-uptake region was carried out by insertional
mutagenesis, using the transposon Tn3-HoHo1. Insertion of this transposon
causes, in addition to the mutation, -galactosidase operon fusions (Stachel et al.
1985). A collection of mutants generated by this approach was then analysed for
the production of the components of the iron-uptake system, permitting the
definition of six genetic units, as well as the identification of the direction of
transcription (Actis et al., 1988; Tolmasky et al., 1988, 1988a). Genetic units I,
IV and VI are only involved in production of anguibactin. Genetic unit V was
recently shown to be an insertion sequence and it seems not to participate in the
biosynthesis of anguibactin or components of the receptor complex (Tolmasky
and Crosa, 1995). A gene encoding a histidine decarboxylase enzyme essential
for biosynthesis of anguibactin has been characterized in genetic unit VI
(Tolmasky et al., 1995). Another gene, essential for synthesis of 2,3-dihydro-
xybenzoic acid, a precursor of anguibactin, was found in the chromosome of V.
anguillarum (Chen et al., 1994). Genetic unit II carries the transport genes fatA,
fatB, fatC and fatD, and genetic unit III carries the angR gene. This latter gene
encodes the protein AngR, which has dual functions: it is a regulator of
expression of other genes (Salinas et al., 1989; Farrell et al., 1990) and can also
complement an E. coli mutant that cannot produce the enzyme 2,3-
dihydroxybenzoateadenosine monophosphate (AMP) ligase (EntE),
suggesting that it may be involved in the biosynthesis of anguibactin (Tolmasky
et al., 1993). A scheme showing the location and orientation of each mutation, as
535 Vibriosis
well as their ability to promote -galactosidase activity, is depicted in Fig. 14.6.
Recent molecular and genetic analysis showed that the regulation of the
expression of this iron-uptake system is indeed complex, involving the
expression of an iron-regulated antisense RNA (Waldbeser and Crosa, 1991;
Salinas et al., 1993) and a protein similar to the Fur repressor originally
described in E. coli (Waldbeser et al., 1993; Tolmasky et al., 1994; Wertheimer
et al., 1994).
Many V. anguillarum strains isolated from various geographical regions
carry plasmids that are highly related, albeit not identical, to pJM1 (Tolmasky
et al., 1985, 1988b). Some of those pJM1-like plasmids, chiefly those present in
strains isolated from the Atlantic Ocean, specified an increased anguibactin
production as compared with the pJM1-mediated anguibactin levels found in
V. anguillarum strains isolated from the Pacific Ocean (Tolmasky et al., 1988b).
The angR gene of one of these pJM1-like plasmids has recently been identified
as the element responsible for the enhanced biosynthesis of anguibactin in the
strain 531A (Tolmasky et al., 1988b, 1993; Salinas et al., 1989).
More recently, polymorphisms in pJM1-like plasmids were also observed in
several other V. anguillarum strains belonging to serotype O1 (Wiik et al.,
1989a,b; Olsen and Larsen, 1990).
Several pathogenic plasmidless V. anguillarum strains belonging to serotype
O1 and O2 have recently been isolated. All these strains were shown to possess a
chromosomally encoded iron-uptake system (Lemos et al., 1988; Conchas et al.,
1991). Experiments using DNA hybridization showed that this system is not
related to that encoded by pJM1. Biochemical characterization of the
chromosomally encoded siderophore demonstrated that it belongs to the family
of the catechols and it may be functionally related to enterobactin. Although
Fig. 14.6. Physical and genetic map of mutations generated by insertion of Tn3-HoHo1 in
recombinant clones carrying the pJ M1 iron uptake region. Restriction endonuclease sites are
shown for EcoRI (E) and XhoI (X). Vertical bars show insertions that resulted in silent mutants.
Short stalks indicate location of insertions that impaired the iron uptake system. Insertions in
which the promoterless lacZ in Tn3-HoHo1 is orientated from left to right are above the bar
representing the DNA fragment, and those insertions in which lacZ is orientated from right to left
are indicated below the bar. The large arrows indicate the orientation of lacZ. Open circles
represent insertions with no -galactosidase activity, closed circles indicate that -galactosidase
was produced constitutively and squares represent iron-regulated production of the enzyme.
Numerals I to VI and the distinctive shading symbolize the different genetic units. (From Tolmasky
et al., 1988a, with permission from the authors and the American Society for Microbiology.)
536
L.A. Actis et al.
several iron-regulated outer membrane proteins were detected by SDS-
polyacrylamide gel electrophoresis (PAGE), none of them have yet been shown
to be involved in the iron-uptake process mediated by this chromosomally
encoded siderophore.
Recently, it was reported that V. anguillarum strains can obtain iron from
other sources, such as haem, haemoglobin and haptoglobinhaemoglobin,
regardless of the type of siderophore produced (Mazoy and Lemos, 1991).
Binding assays and Western blots showed the presence of specific receptors for
heme groups in the cell membrane of V. anguillarum (Mazoy and Lemos, 1996).
Some of these haem-binding proteins are iron-regulated while others are
expressed independently of the iron status of the cell. The significance of this
siderophore-independent iron-acquisition system in virulence is still unknown.
The production of a protease, a potential virulence factor, has been
described by Norqvist et al. (1990) and Farrell and Crosa (1991). An elastolytic
metalloprotease of 36 kDa, requiring Zn
+2
for its activity and Ca
+2
for its stability
was found to be associated with the invasion properties of the V. anguillarum
NB10 strain. Virulence studies using proteolytic activity mutants showed that
they have a 1000-fold higher median lethal dose (LD
50
) when assayed by
immersion and a tenfold higher LD
50
when assayed by intraperitoneal injection
(Norqvist et al., 1990). In addition, Farrell and Crosa (1991) purified a
metalloprotease from another V. anguillarum strain, 514, which seems to be
either related or identical to that described in strain NB10. The molecular mass
of this purified protease was calculated to be 38 kDa. In this case, the protease
activity was ethylenediaminetetra-acetic acid (EDTA)-sensitive and it could be
restored by the addition of both Ca
+2
and Zn
2+
. Biochemical analysis as well as
determination of the N-terminal amino acid sequence showed that the
V. anguillarum protease(s) are highly related to proteases already described in
other Vibrio species, such as V. cholerae and V. vulnificus, as well as to the
elastase of Pseudomonas aeruginosa and the protease of Legionella
pneumophila (Norqvist et al., 1990; Farrell and Crosa, 1991).
It has been demonstrated that fish sera can kill strains of V. anguillarum.
Thermal tolerance and treatment with either EDTA or EGTA demonstrate that
the mechanism for bacterial killing by rainbow trout serum is the alternate
complement pathway (Trust et al., 1981). Resistance to this bactericidal action
of normal non-immune serum seems to contribute to the virulence of pathogenic
V. anguillarum (Trust et al., 1981; Toranzo et al., 1983a,b). Pathogenic strains of
V. anguillarum isolated from striped bass (Morone saxatilis) were resistant to the
lytic activity of unheated fish serum, whereas other non-pathogenic
V. anguillarum strains were sensitive, indicating that this factor may play an
important role in the virulence properties of this bacterium (Toranzo et al.,
1983a). Strains in which the pJM1 plasmid was cured were still resistant to the
bactericidal action of the serum, demonstrating that this trait is encoded by the
by V. anguillarum 775 chromosome rather than the pJM1 plasmid (Trust et al.,
1981).
Haemagglutinating activity is another bacterial factor that is believed to play
a central role in the infectivity of bacterial pathogens. It was found that
pathogenic V. anguillarum strains isolated from the US east coast produced
537 Vibriosis
strong haemagglutinins for fish erythrocytes, while this activity was not detected
in non-virulent strains (Toranzo et al., 1983b). The pathogenic strains showed
two agglutination patterns, depending on the type of erythrocytes agglutinated.
This agglutination activity was inhibited by D-mannose, indicating that this
carbohydrate or an analogue is part of the erythrocyte receptor for this
haemagglutinin. Although a haemagglutinating activity was also detected in
V. anguillarum strains isolated from the Pacific North-West, no correlation
between the expression of this activity and the strain virulence could be
established (Trust et al., 1981).
The secretion of a V. anguillarum extracellular toxin was reported by
Kodama et al. (1985). This toxin was purified by gel-filtration chromatography,
and molecular characterization of the active fraction revealed that the toxic
activity was associated with two 44-kDa proteins and a single 34-kDa protein.
These proteins were lethal to rainbow trout and mice when injected
intraperitoneally and intravenously, respectively. This toxic activity affected the
intestinal tract and the vascular system of the injected animals, and could be
neutralized by a specific antiserum raised in rabbit. Chemical analysis of these
components indicated that some of them are associated with carbohydrates. The
toxic activity was sensitive to heating and potassium periodate, and resistant to
trypsin and acetone treatments. Furthermore, the toxin was not related to either
haemolytic or protease activities. This exocellular toxin is another important
virulence factor in vibrios since its haemorrhagic, cytotoxic and lethal effects
could lead to the pathological alterations observed in infected fish. In another
study, Aoki et al. (1985) found that a V. anguillarum strain became more virulent
after passages in ayu, and the increase of virulence was correlated with an
increase in the amount of production of its lipopolysaccharide: Furthermore, the
increase in virulence was also correlated with a change in the size of the
lipopolysaccharide: the virulent strain had a high-molecular weight component
that was absent in the low-virulence strain.
It has been suggested that there is a correlation between the virulence of
V. anguillarum and the presence of more than one flagellum (Chart, 1983;
Norqvist and Wolf-Watz, 1993). In a recent study, Milton et al. (1996) cloned
and characterized a V. anguillarum flagellin gene (flaA). Mutations in this gene
resulted in partially motile bacteria. Virulence immersion studies concluded that
the product of the flaA gene is needed for crossing the fish integument and may
play a role in virulence (Milton et al., 1996).
Transposition mutagenesis revealed the presence of two genes, virA and
virB, encoding a major surface antigen important for the virulence of
V. anguillarum (Norqvist and Wolf-Watz, 1993). This surface antigen appears to
be a lipopolysaccharide located on the outer sheath of the flagellum and is
expressed in vivo during fish infections together with the flagellum. Recently,
Milton et al. (1994) identified virC, another gene that is essential for virulence in
V. anguillarum. Although mutations in this gene result in strains that lose
virulence, the function of the product of virC is still unknown.
538
L.A. Actis et al.
Vibrio ordalii
The bacterium
Vibrio ordalii is another major cause of vibriosis in wild and cultured marine
salmonids in the Pacific North-West of USA and Japan. Vibrio ordalii is a
polarly flagellated, Gram-negative, curved rod that has been reported as Vibrio
sp. 1669 (Harrel et al., 1976), Vibrio sp. RT (Ohnishi and Muroga, 1976), V.
anguillarum biotype 2 (Schiewe et al., 1977), B. anguillara biotype II (Baumann
et al., 1978), but later amended to V. anguillarum biotype II, and V. anguillarum
phenon II, until its taxonomical status was clarified by Schiewe et al. (1981).
This bacterium is phenotypically and genetically distinct from V. anguillarum
(Schiewe et al., 1981), and it has been isolated from natural infections in coho
salmon (Oncorhynchus kisutch), as well as from experimental infections in
chum salmon and spring chinook salmon (Oncorhynchus tshawytscha) (Ransom
et al., 1984). It could be cultivated at 30C in common media, such as trypticase
soy broth or brain heart-infusion, with the addition of 1% NaCl. The biochemical
characteristics of V. ordalii are reported elsewhere (Schiewe et al., 1981). Table
14.2 lists the biochemical tests frequently used to distinguish between V. ordalii
and V. anguillarum. Vibrio ordalii has a reduced nutritional and physiological
versatility, depending heavily on the host to survive and consequently V. ordalii
has only been isolated from moribund fish. Schiewe et al. (1981) performed
DNA analysis of a number of strains of this bacterium. Their findings showed
that the mol % G + C content of this bacterium DNA is 4344%, and
demonstrated that isolates of V. ordalii have more than 83% homology within the
group, while showing only 5869% homology to V. anguillarum. There was
little or no homology with DNA isolated from Vibrio parahaemolyticus, Vibrio
alginolyticus and other unclassified vibrios.
The molecular characterization of different isolates of V. ordalii revealed the
presence of a high-copy-number plasmid in all the strains examined (Schiewe
Table 14.2. Phenotypic properties used to differentiate Vibrio anguillarum
from V. ordalii (data from Schiewe et al., 1981).
Biochemical tests and
growth temperature V. anguillarum V. ordalii
Argininealkaline reaction +
Citrate, Christensen +
Citrate, Simmons +
Lipase +
ONPG +
Starch hydrolysis +
VogesProskauer +
Acid production from
Cellobiose +
Glycerol +
Sorbitol +
Trehalose +
Growth at 37C +
OPNG, o-nitrophenyl -D-galactopyranoside.
539 Vibriosis
and Crosa, 1981). This cryptic plasmid, designated pMJ101, is a 30-kb
extrachromosomal element that is not related to the pJM1 virulence plasmid
present in V. anguillarum 775, and does not share homology with other plasmids
belonging to already defined incompatibility groups. Recent work initiated the
characterization of this plasmid at the molecular level (Bidinost et al., 1994). A
restriction map was obtained, and cloning and transformation experiments
allowed the localization and isolation of the replication region of pMJ101. By
using site-directed and Tn5 transposition mutagenesis, in combination with
subcloning experiments, the essential replication functions were localized
within a 2.4 kb EcoRVHindIII restriction fragment (Fig. 14.7). Recombinant
clones carrying this fragment were able to replicate in E. coli cells deficient in
DNA polymerase 1 (PolI) or integration host factors (IHF). However, the
replication of plasmid derivatives containing the pMJ101 ori region was highly
dependent on the presence of a functional dam methylase, suggesting that
methylation is a requirement for the replication of this plasmid. The ability of
pMJ101 derivatives to replicate in the absence of PolI suggested that this
plasmid encodes its own replication protein. Electrophoretic analysis of radio-
labelled plasmid-encoded proteins showed that a 36 kDa protein is encoded
within the replication region and is essential for the replication of pMJ101. In
spite of this analysis, the role of pMJ101 in the V. ordalii life cycle and/or
virulence remains unknown. Therefore, the isolation of a derivative with no
plasmid will be an invaluable tool to assess the function of this universal plasmid
present in V. ordalii isolates.
The disease
Histopathological studies in naturally acquired vibriosis in chum salmon
showed that V. ordalii has a different tissue tropism compared with
V. anguillarum, since it localizes more frequently in the muscle and skin, with
bacterial colonies or aggregations that can replace large areas of host tissues
(Ransom, 1978; Schiewe, 1983). In some of the infected places, the bacterium
Fig. 14.7. Physical and genetic map of pMJ 101. Restriction endonuclease sites are shown for
BamHI (B), BglII (Bg), HindIII (H), EcoRI (EI), EcoRV (EV), and XbaI (X). The DNA fragment
harbouring the minimal pMJ 101 replication region is indicated by the shaded area.
540
L.A. Actis et al.
also causes the necrosis and haemorrhaging of the surrounding tissues. These
findings indicate that the bacterium can enter the host by invasion of the
salmonid integument. Colonies of V. ordalii are commonly found in loose
connective tissues in the gills, throughout the digestive tract and in the pyloric
caeca, suggesting that the infection could also begin at these sites. Occasionally,
V. ordalii can be observed as microcolonies in spleen and liver, and low counts in
blood may be observed during the initial stages of the infection. A similar
pathology was observed when either chum, coho or chinook salmons were
exposed to a large number of bacterial cells in experimental water borne
infections. This observation established that this is a valid model to study the
mechanisms of pathogenesis, as well as the bacterial virulence factors involved.
Juvenile salmon exposed to V. ordalii by parenteral challenge developed a
systemic infection and the bacterium was recovered from liver, kidneys, spleen
and blood immediately after the infection (Schiewe, 1983). However, the
number of bacteria in the liver declined after 1 h and then increased 22 h
postinfection, bacterial numbers were high in all the organs and 100% mortality
occurred 6 days after infection.
Virulence factors
During the progression of the infection, there is a correlation between the
presence of V. ordalii in the host blood and a marked decrease in white blood cell
counts in moribund fish (Harbell et al., 1979; Ransom et al., 1984), suggesting
the production of a leucocytolytic factor. This factor may play an important role
in the pathogenesis of the infection.
Other factors can also play an important role in pathogenesis, such as the
ability of this bacterium to resist the bactericidal activity of normal non-immune
rainbow trout serum, which has been correlated with the virulence of this
bacterium (Trust et al., 1981). It was also reported that V. ordalii strains can
agglutinate trout and human erythrocytes, as well as yeast cells, although some
discrepancies were reported, probably due to technical artefacts (Trust et al.,
1981; Larsen and Mellergaard, 1984). The ability of V. ordalii to agglutinate
different eukaryotic cell types suggests that it could attach to and interact with
the host cells. However, a correlation between the agglutination phenotype and
the virulence properties of the strains studied could not be obtained, and it
remains to be proved whether these agglutinins play a role during the onset and
progress of infections in salmonids.
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 0.54% NaCl with an
optimum salt concentration of 1.5% (Egidius et al., 1981). The bacterium is
541 Vibriosis
psychrophilic and can grow between 1C and 22C, with an optimum
temperature of 1215C. 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
Fig. 14.8. Electron micrograph of Vibrio salmonicida showing a polar tuft of nine sheathed
flagella. 7120. Bar =1 m. (From Egidius et al., 1986, with permission from the authors and the
American Society for Microbiology.)
542
L.A. Actis et al.
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
(Srum 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
(Srum 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
DNADNA 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 (Srum et al., 1990). No correlation could be
found between the plasmid profiles and the LD
50
values of the strains analysed.
Valla et al. (1992) has analysed the 10 MDa plasmid pVS1 present in the strain
TEO83.00. 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 TEO83.001.
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 Srum et al., 1990).
Geographical region of Norwegian coast*
Plasmid pattern
(MDa) 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.
543 Vibriosis
derivative demonstrated that there is not a significant correlation between the
plasmid content and the virulence of V. salmonicida TEO83.001. 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 (Srum 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 (Srum 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 (Srum 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.
The disease
Hitra disease appeared in 1977 and occurred for the first time on a large scale in
1979 in fish farms in the Norwegian island of Hitra. Since then, it has devastated
fish farms located along the western and northern Norwegian coast (Egidius et
al., 1981). However, single outbreaks have also been reported in Scotland
(Bruno et al., 1985, 1986), on the Faroe Islands (Dalsgaard et al., 1988) and in
New Brunswick and Nova Scotia, Canada (referenced in Srum et al.,1992).
This disease affects mainly fish farms with Atlantic salmon and occasionally
with rainbow trout. Vibrio salmonicida has also been described as the
aetiological agent of cold-water vibriosis affecting farmed cod in Norway
(Srum et al., 1990). Hitra disease occurs mainly in late autumn, winter or early
spring (Egidius et al., 1981). The characteristics of this disease, also known as
haemorrhagic syndrome, are anaemia and haemorrhages with a generalized
septicaemia, presenting large amounts of bacterial cells in the blood of moribund
or recently dead fish. The haemorrhages are mainly found in the integument
surrounding the internal organs of the fish (Poppe et al., 1985; Egidius et al.,
1986). However, in the recently described cases of cod infections, some
differences were found in the pathology. The infected cod fry show cataracts,
cranial haemorrhage and splenomegaly, symptoms that are closer to those
observed in cod when infected with V. anguillarum (referenced in Srum et al.,
1990).
544
L.A. Actis et al.
Other vibrios
Although all three vibrios already described are the most studied agents of
vibriosis, other species have also been identified, some of them being also
pathogenic to humans.
Vibrio damsela was identified as the causative agent of skin ulcers present in
diseased blacksmith, a temperate-water damselfish (Chromis punctipinnis)
(Love et al., 1981). The ulcers vary from 0.5 to 2 cm in diameter and are
characterized by muscle lysis and histiocytes present in the dermis and skeletal
muscles (Love et al., 1981). Experimental infections, in which wounds were
artificially induced and V. damsela cells were swabbed, showed that ulcers
appeared after 3 days and the animals died on the fourth day. In other
experiments, V. damsela was shown to induce ulcer formation by directly
swabbing the flank, although in this case only 50% of the experimental animals
died. Infections with V. damsela seem to be limited to blacksmith populations,
between August and October, on the Californian coast, and from June to August
on Cataline Island (references in Love et al., 1981). Outbreaks may be due to
elevated water temperature and growth of the bacteria. Another factor
contributing to the outbreaks could be a reduction of host defences in
blacksmiths during late summer, when they are breeding (Love et al., 1981).
The pathogenicity of V. damsela is correlated with production of a cytolysin,
named damselysin, using mice as the animal model (Kreger, 1984). This
cytolysin was purified and identified as a phospholipase D (Kothary and Kreger,
1985; Kreger et al., 1987). The gene encoding damselysin was recently cloned
from the chromosome of V. damsela and expressed in E. coli (Cutter and Kreger,
1990). The recombinant clone containing this gene, designated dly, was used as a
probe in dot-blot hybridizations against different species of the genus Vibrio. No
homology was detected in these assays. In addition, these studies demonstrated
that highly haemolytic strains of V. damsela showed homology to the dly gene,
while those strains that had lower haemolytic activity had no homology to this
gene. These authors also demonstrated, using Southern blot hybridization
analysis with the same probe against restriction endonuclease-treated genomic
DNA, that two different fragments had homology to the dly probe. The fragment
detected in hybridizations against DNA from the intermediate to low haemolytic
strains was larger than that detected using DNA from highly haemolytic strains.
The authors suggested that this was an indication of a damselysin gene
rearrangement that could be responsible for the different haemolytic activities
detected in these strains (Cutter and Kreger, 1990).
However, it has been suggested that several other factors contribute to the
virulence of V. damsela. It was recently shown that the ability to resist the
bactericidal effect of non-immune serum correlates with virulence, since only
virulent strains were able to grow in the presence of untreated human and turbot
sera (Fouz et al., 1994). Furthermore, this report showed that the growth of this
bacterium was enhanced by the addition of haemoglobin and ferric ammonium
citrate. These results indicate that the ability to acquire iron may play a central
role in the pathogenesis of the infections caused by this fish pathogen.
Vibrio damsela was also identified as a human pathogen; several cases were
545 Vibriosis
reported of severe, progressive necrotizing infection found in wounds of patients
living in coastal regions (Morris et al., 1982; Coffey et al., 1986).
Vibrio vulnificus, also known as Vibrio anguillicida, is capable of causing
disease in cultured eels (Anguilla japonica) (Nishibuchi et al., 1979, 1980). It
was isolated during outbreaks of vibriosis in eels in Japan (Muroga et al., 1976)
and the UK (Austin, 1987). This fish pathogen can be identified using an ELISA
for the haemolysin, avoiding the lengthy and labour-intensive biochemical
assays used for its identification (Parker and Lewis, 1995). Recently, a nested
polymerase chain reaction (PCR) method was developed for rapid and sensitive
detection of V. vulnificus in fish and environmental specimens (Arias et al.,
1995), using rRNA-targeted oligonucleotide probes specific for V. vulnificus
(Aznar et al., 1994). The comparison among clinical, environmental and eel
isolates of V. vulnificus showed that, although highly related, the eel isolates
have distinct phenotypic, cultural and serological properties. Furthermore, only
the eel isolates were pathogenic to eels, as well as to mice, while typical V.
vulnificus isolates were only pathogenic to mice (Tison et al., 1982). These
differences indicated that the eel isolates belong to a different biogroup, and it
was thus proposed that the V. vulnificus strains similar to those isolated from
diseased eels should be classified as V. vulnificus biogroup 2, keeping the
designation of V. vulnificus biogroup 1 for clinical and environmental strains
(Tison et al., 1982).
Vibrio vulnificus biogroup 2 survives in brackish water and attaches to eel
surfaces, suggesting that water and infected fish act as reservoirs (Amaro et al.,
1995). In addition, the survival of this bacterium in the environment and the
spread of the disease depend on the temperature and salinity of the water (Kaspar
and Tamplin, 1993; Amaro et al., 1995).
The virulence of this salt-requiring bacterium is associated with bacterial
phenotypic properties such as colony morphology and presence of capsule
(Yoshida et al., 1985; Simpson et al., 1987; Wright et al., 1990; Amaro et al,
1994, 1995). It was suggested that the production of a capsule by V. vulnificus
biotype 2 is essential for the survival of the pathogen in the marine environment
and the colonization and invasion of the host. This conclusion is based on the
fact that non-encapsulated cells can cause vibriosis in eels when injected
intraperitoneally, although pure cultures of encapsulated cells were always
recovered from diseased fish (Biosca et al., 1993). Other potential virulence
factors include the secretion of extracellular cytotoxic and cytolytic factors
(Kreger and Lookwood, 1981; Gray and Kreger, 1985; Amaro et al., 1994), an
elastolytic protease (Kothary and Kreger, 1987), an extracellular phospholipase
(Testa et al., 1984), resistance to phagocytosis and the bactericidal effects of sera
(Johnson et al., 1984; Yoshida et al., 1985). The availability of iron was also
correlated with the virulence of V. vulnificus (Wright et al., 1981; Jayalakshmi
and Venugopalan, 1992; Amaro et al., 1994). Clinical and environmental isolates
of V. vulnificus can use haem-containing compounds, such as haemoglobin,
haemoglobinhaptoglobin, haemin, and haeminalbumin to reverse iron
limitation (Zakaria-Meehan et al., 1988; Nishina et al., 1992; Simpson and
Oliver, 1993). However, the utilization of haemoglobinhaptoglobin and
haeminalbumin is correlated with the expression of a proteolytic activity that is
546
L.A. Actis et al.
positively regulated by the presence of either haemin or haemoglobin in the
medium (Simpson and Oliver, 1993). Simpson and Oliver (1983) showed that
this bacterium secretes iron-chelating compounds when cultured under iron
deficiency. Recently, a new siderophore, named vulnibactin, was purified and
characterized as a molecule containing 2,3-dihydroxybenzoic acid, salicylic
acid, L-threonine and norspermidine (Okujo et al., 1994).
Severe human infections caused by V. vulnificus biogroup 1 have been
reported (Blake et al., 1980), and environmental studies demonstrated that this
bacterium is widespread along the coasts (Oliver et al., 1983). A recent case of
human infection caused by a V. vulnificus strain very similar to the eel isolates
was described (Veenstra et al., 1992). Although this human isolate was indole-
negative, the main characteristic of biogroup 2, other reactions, such as the
ornithine decarboxylase test, and growth at 42C were different from those
described by Tison et al. (1982). The infection seemed to be caused from contact
with an infected eel through an open wound (Veenstra et al., 1992). It was
recently reported that V. vulnificus fatal infections are also associated with eating
contaminated raw or undercooked seafood, particularly raw oysters (Mascola et
al., 1996). This study demonstrated that immunocompromised patients and
persons with chronic liver diseases are at increased risk.
Vibrio pelagius and V. splendidus have recently been described as new
bacteria causing vibriosis in juvenile and adult turbot (Lupiani et al., 1989).
Pathogenicity of V. splendidus was confirmed by experimental infections
(Toranzo and Barja, 1990). However, most if not all the virulence mechanisms of
these fish pathogens are still unknown, and further investigations are essential
not only to elucidate these mechanisms but also to identify essential bacterial
products that could lead to the development of more efficient prophylactic and
treatment methods for vibriosis.
ACKNOWLEDGEMENTS
We are grateful to Dr Michael Schiewe for providing the photograph of the
diseased fish shown in Fig. 14.2. The experiments from Dr Crosas laboratory
reported in this work were supported by Public Health Service Grant No. 19018
from the National Institutes of Health awarded to Dr Jorge H. Crosa.
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